Inhibiting the transient receptor potential a1 ion channel

ABSTRACT

The present invention relates to compounds of the Formula (I), or a pharmaceutically acceptable salt, pharmaceutical preparation, or pharmaceutical composition thereof, and their use for the treatment of pain, inflammatory disease, neuropathy, dermatological disorders, pulmonary conditions, and cough, as well as inhibiting the Transient Receptor Potential A1 ion channel (TRPA1).

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Application No.61/983,223, filed Apr. 23, 2014, and U.S. Provisional Application No.61/987,272, filed May 1, 2014, both of which are herein incorporated byreference in their entirety.

TECHNICAL FIELD

The present invention relates to compounds of the Formula (I), or apharmaceutically acceptable salt, pharmaceutical preparation, orpharmaceutical composition thereof, and their use for the treatment ofpain, inflammatory disease, neuropathy, dermatological disorders,pulmonary conditions, and cough, as well as inhibiting the TransientReceptor Potential A1 ion channel (TRPA1).

BACKGROUND

Transient Receptor Potential A1 (herein, “TRPA1”) is a non-selectivecation channel related to pain sensation in humans. TRPA1 is found insensory neurons and functions as a detector that helps link detection ofnoxious chemicals, tissue damage, and inflammation to pain. Activationof TRPA1 is believed to cause pain by inducing firing of nociceptiveneurons and driving central sensitization in the spinal cord. TRPA1stimulation can also increase firing of sensory neurons, leading to therelease of pro-inflammatory neuropeptides such as NK-A, substance P andCGRP, which induce vasodilation and help recruit immune cells. A varietyof endogenous reactive compounds produced during inflammation activateTRPA1, including 4-hydroxynonenal released during liposome peroxidation;cyclopentane prostaglandins synthesized by COX enzymes; hydrogenperoxide produced by oxidative stress. Activation of TRPA1 alsosensitizes TRPA1 to cold. Furthermore, a gain-of-function mutation inTRPA1 causes familial episodic pain syndrome; patients suffering fromthis condition have episodic pain that may be triggered by cold. Thus,TRPA1 is considered to play a role in pain related to nerve damage, coldallodynia, and inflammatory pain.

Compounds that inhibit the TRPA1 ion channel can be useful, for example,in treating conditions ameliorated, eliminated, or prevented byinhibition of the TRPA1 ion channel. For example, pharmaceuticalcompositions that inhibit TRPA1 can be used to treat pain. Inhibition ofTRPA1 (e.g., by genetic ablation and chemical antagonism) has been shownto result in reduced pain behavior in mice and rats. Knockout micelacking functional TRPA1 have diminished nociceptive responses to TRPA1activators, including AITC, formalin, acrolein, 4-hydroxynonenal, and,in addition, have greatly reduced thermal and mechanicalhypersensitivity in response to the inflammatory mediator bradykinin(e.g., Kwan, K. Y. et al. Neuron 2006, 50, 277-289; Bautista, D. M. etal. Cell 2006, 124, 1269-1282). In animal pain models, down regulationof TRPA1 expression by gene specific antisenses prevented and reversedcold hyperalgesia induced by inflammation and nerve injury (see, e.g.,Obata, K. et al., J Clin Invest (2005) 115, 2393-2401; Jordt, S. E. etal., Nature (2004), 427, 260-265; Katsura, H. et al., Explor Neurol(2006), 200, 112-123). TRPA1 inhibitor compounds are effective in avariety of rodent pain models. TRPA1 inhibitors have been shown toreduce mechanical hypersensitivity and cold allodynia followinginflammation induced by Complete Freund's Adjuvant without alteringnormal cold sensation in naïve animals and also to improve function inthe rat mono-iodoacetate osteoarthritis model (see, e.g., Materazzi, Set al., Eur J Physiol (2012), 463(4):561-9; Wei H et al., Anesthesiology2012, 117(1):137-48; Koivisto, A et al., Pharmacol Res (2012),65(1):149-58). TRPA1 inhibitor compounds have demonstrated reduced painbehavior in rodents injected with AITC (mustard oil), formalin,cinnamaldehyde, acrolein, and other TRPA1 activators. TRPA1 inhibitorcompounds have also demonstrated efficacy in rodent models for postoperative pain, (see, e.g., Wei et al., Anesthesiology (2012),117(1):137-48); chemotherapy induced peripheral neuropathy (see, e.g.,Trevisan, et al., Cancer Res (2013) 73(10):3120-31), and painfuldiabetic neuropathy (see, e.g., Koivisto et al., Phannacol Res (2011)65:149-158).

SUMMARY

The compounds described herein can be useful in the treatment ofdisorders wherein inhibition of the TRPA1 ion channel is beneficial, forexample, in the treatment of pain. In some embodiments, a compounddescribed herein has preferable properties over other compounds in theart that inhibit TRPA1. For example, in some embodiments, a compounddescribed herein inhibits the TRPA1 ion channel without elevating serumbiomarkers of hepatotoxicity. In some embodiments, a compound asdescribed herein, e.g., a compound of Formula (I), has desirable aqueoussolubility (including compounds with aqueous solubility suitable forpharmaceutical compositions formulated for intravenous administration)relative to other compounds in the art that inhibit TRPA1.

Described herein is a compound of the Formula (I) and pharmaceuticallyacceptable salts thereof:

wherein each of the variables above are as described herein, forexample, in the detailed description below.

Also described herein are purified pharmaceutical preparations andpharmaceutical compositions comprising a compound of Formula (I) or apharmaceutical salt thereof.

The compounds and compositions described herein can be used to treatvarious disorders in a subject. For example, described herein aremethods of treatment such as a method of treating a TRPA1 mediateddisorder in a subject, the method comprising administering an effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt thereof. Methods of treating pain in a subject, the methodcomprising administering an effective amount of a compound of Formula(I), or a pharmaceutically acceptable salt thereof are also describedherein. Exemplary types of pain include neuropathic pain, e.g., painfuldiabetic neuropathy, chemotherapy-induced peripheral neuropathy, lowerback pain, trigeminal neuralgia, post-herpetic neuralgia, sciatica, andcomplex regional pain syndrome; inflammatory pain, e.g., from rheumatoidarthritis, osteoarthritis, temperomandibular disorder; PDN or CIPN;visceral pain, e.g., from pancreatitis, inflammatory bowel disease,colitis, Crohn's disease, endometriosis, pelvic pain, and angina; painselected from the group: cancer pain, burn pain, oral pain, crush andinjury-induced pain, incisional pain, bone pain, sickle cell diseasepain, fibromyalgia and musculoskeletal pain; or pain from hyperalgesiaor allodynia.

In some embodiments the methods include treating inflammatory disease ina subject, the method comprising administering an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

In some embodiments the methods include treating neuropathy in asubject, the method comprising administering an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.In some embodiments, the neuropathy is from diabetes, chemical injury,chemotherapy, and or trauma.

In some embodiments the methods include treating a dermatogologicaldisorder in a subject, the method comprising administering an effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt thereof. Exemplary dermatogological disorders include atopicdermatitis, acute pruritus, psoriasis, hives, eczema, dyshidroticeczema, mouth ulcers, and diaper rash.

In some embodiments the methods include treating a pulmonary conditionin a subject, the method comprising administering an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. Exemplary pulmonary conditions include obstructive diseasessuch as chronic obstructive pulmonary disease. Additional exemplarypulmonary conditions include asthma and cough.

In addition, a compound as described herein, e.g., a compound of Formula(I), are useful in the manufacture of a pharmaceutical compositionformulated for oral administration. In some embodiments, a compounddescribed herein can be formulated into a composition for intravenousadministration. In embodiments, a compound or composition describedherein can be used to treat pain

A compound as described herein, e.g., a compound of Formula (I), caninclude molecules having one or more chiral centers. For example, unlessotherwise stated, a composition of Formula (I) can contain variousamounts of stereoisomers of Formula (Ia), (Ib), (IIa) and (IIb). In anembodiment, a composition comprising a compound of Formula (Ia) or (IIa)preferably contains a therapeutically effective amount of the compoundhaving the stereochemistry indicated in Formula (Ia) or (IIa) (e.g., anenantiomeric excess or a diastereomeric excess of a particular isomer ofFormula (Ia) or (IIa) over the corresponding stereoisomer of Formula(Ib) or (IIb)). In an embodiment, a composition comprising a compound ofFormula (I) contains a therapeutically effective amount of the compoundhaving the stereochemistry indicated in Formula (Ib) or (IIb) (e.g., anenantiomeric excess or a diastereomeric excess of a particular isomer ofFormula (Ib) or (IIb) over the corresponding stereoisomer of Formula(Ia)).

In addition, compounds of Formula (I) can include one or more isotopesof the atoms present in Formula (I). For example, compounds of Formula(I) can include: those in which H (or hydrogen) is replaced with anyisotopic form of hydrogen including ¹H, ²H or D (Deuterium), and ³H(Tritium); those in which C is replaced with any isotopic form of carbonincluding ¹²C, ¹³C, and ¹⁴C; those in which O is replaced with anyisotopic form of oxygen including ¹⁶O, ¹⁷O and ¹⁸O; those in which N isreplaced with any isotopic form of nitrogen including ¹³N, ¹⁴N and ¹⁵N;those in which P is replaced with any isotopic form of phosphorousincluding ³¹P and ³²P; those in which S is replaced with any isotopicform of sulfur including ³²S and ³⁵S; those in which F is replaced withany isotopic form of fluorine including ¹⁹F and ¹⁸F; and the like. In anembodiment, compounds represented by Formula (I) comprise isomers of theatoms therein in their naturally occurring abundance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a spectrum depicting the X-ray powder diffraction (XRPD)pattern of a solid crystalline form of Compound 2 (Form A) after slurrytreatment in ethanol.

FIG. 2 is a spectrum depicting the X-ray powder diffraction pattern ofan anhydrous solid crystalline form of Compound 2 (Form B) after slurrytreatment in 97% ethanol/3% water and drying under vacuum (−80° C. forone day).

FIG. 3 is a graph depicting the results of differential scanningcalorimetry (DSC) analysis on an anhydrous solid crystalline form ofCompound 2 (Form B).

FIG. 4 is a graph depicting the results of thermal gravimetric analysis(TGA) on an anhydrous solid crystalline form of Compound 2 (Form B).

FIG. 5 is a graph depicting the results of dynamic vapor sorption (DVS)analysis on an anhydrous solid crystalline form of Compound 2 (Form B).

FIG. 6 is a spectrum depicting the overlaid results of XRPD analysis ofthe anhydrous solid crystalline form of Compound 2 (Form B) before(light gray trace) and after (dark gray trace) microionization to a d₉₀value of less than 10 microns.

FIG. 7 is a graph depicting the effect of varying dosage amounts ofCompound 2 administered orally in the CFA-induced cold hyperalgesiamodel in the rat.

FIG. 8 is a chart depicting the duration of formalin-mediated painbehaviors post oral administration of Compound 2. Compound 2 was dosedat 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or 24hours prior to formalin injection to assess the persistence of thebenefit provided by treatment.

FIG. 9 is a chart depicting an exemplary profile of CYP450 reactionphenotyping with Compound 2, also referred to herein as Example 2.

FIG. 10 is a graph depicting the solubility of a micronized formulationof Compound 2 over the pH range of 2.00 to 8.00.

FIG. 11 is a graph depicting plasma levels of Compound 2 (i.e., Example2) in rat, dog, or monkey models after administration of a 10 mg/kg oraldose.

FIG. 12 is a graph depicting a comparison of the pharmacokinetic profileof Compound 2 (i.e., Example 2) in capsule and suspension formulationsin fed and fasted monkeys.

FIG. 13 is a chart depicting the analgesic effects observed upon lowdoses of orally administered Compound 2 (i.e., Example 2) and a control(Compound A, i.e., Comparator A) in the CFA model.

FIG. 14 is a chart depicting the dose response observed upon oraladministration of Compound 2 (i.e., Example 2) in the formalin model.

FIG. 15 is a chart depicting the efficacy observed with doses ofintravenously administered Compound 1 (i.e., Example 1) in the formalinmodel.

FIG. 16 is a graph depicting the change in lung resistance (early andlate asthmatic response) in sheep challenged with allergen afteradministration of Compound 2.

FIG. 17 is a chart depicting the effect of Compound 2 (i.e., Example 2)on measurement of airway hyperresponsiveness in the sheep model ofallergic asthma.

FIG. 18 is a chart depicting the serum biomarkers of hepatotoxicity inbeagle dogs before and after receiving a once daily oral dose ofCompound 2 over 5 days.

FIG. 19 is a chart depicting the change in serum biomarkers ofhepatotoxicity between a control and Compound 2 (orally administered) inbeagle dogs on Day 5 after receiving a once daily oral dose of Compound2 over 5 days.

FIG. 20 is a chart depicting the change in serum biomarkers ofhepatotoxicity in sprague-dawley rats after receiving a once daily oraldose of Compound 2 over 28 days.

FIG. 21 is a chart depicting the change in serum biomarkers ofhepatotoxicity between a control and Compound 2 (orally administered) insprague-dawley rats on day 28 after receiving a once daily oral dose ofCompound 2 over 28 days.

FIG. 22 is a chart depicting the serum biomarkers of hepatotoxicity incynomolgus monkeys after receiving a once daily oral dose of Compound 2over 28 days.

FIG. 23 is a chart depicting the change in serum biomarkers ofhepatotoxicity between a control and Compound 2 (orally administered) incynomolgus monkeys on day 28 after receiving a once daily oral dose ofCompound 2 over 28 days.

DETAILED DESCRIPTION Definitions

This disclosure is not limited in its application to the details of themethods and compositions described herein. Also, the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

As used herein, an amount of a compound or combination effective totreat a disorder (e.g., a disorder as described herein),“therapeutically effective amount”, “effective amount” or “effectivecourse” refers to an amount of the compound or combination which iseffective, upon single or multiple dose administration(s) to a subject,in treating a subject, or in curing, alleviating, relieving or improvinga subject with a disorder (e.g., a disorder as described herein) beyondthat expected in the absence of such treatment.

The term “pharmaceutically acceptable,” as used herein, refers to acompound or carrier (e.g., excipient) that may be administered to asubject, together with a compound described herein (e.g., a compound ofFormula (I)), and which does not destroy the pharmacological activitythereof and is nontoxic when administered in doses sufficient to delivera therapeutic amount of the compound.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and arethus capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds disclosed herein. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J PharmSci 66:1-19.)

In other cases, the compounds disclosed herein may contain one or moreacidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds disclosed herein. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term, “treat” or “treatment,” as used herein, refers to theapplication or administration of a compound, alone or in combinationwith, an additional agent to a subject, e.g., a subject who has adisorder (e.g., a disorder as described herein), a symptom of adisorder, or a predisposition toward a disorder, with the purpose tocure, heal, alleviate, relieve, alter, remedy, ameliorate, improve oraffect the disorder.

As used herein, the term “subject” is intended to include human andnon-human animals. Exemplary human subjects include a human subjecthaving a disorder, e.g., a disorder described herein. The term“non-human animals” of the invention includes all vertebrates, e.g.,non-mammals (such as chickens, amphibians, reptiles) and mammals, suchas non-human primates, domesticated and/or agriculturally usefulanimals, e.g., sheep, dog, cat, cow, pig, etc.

The terms “antagonist” and “inhibitor” are used interchangeably to referto an agent that decreases or suppresses a biological activity, such asto repress an activity of an ion channel, such as TRPA1. TRPA1inhibitors include inhibitors having any combination of the structuraland/or functional properties disclosed herein.

An “effective amount” of, e.g., a TRPA1 antagonist, with respect to thesubject methods of inhibition or treatment, refers to an amount of theantagonist in a preparation which, when applied as part of a desireddosage regimen brings about a desired clinical or functional result.Without being bound by theory, an effective amount of a TRPA1 antagonistfor use in the methods of the present invention includes an amount of aTRPA1 antagonist effective to decrease one or more in vitro or in vivofunctions of a TRPA1 channel. Exemplary functions include, but are notlimited to, membrane polarization (e.g., an antagonist may preventdepolarization of a cell), ion flux, ion concentration in a cell,outward current, and inward current. Compounds that antagonize TRPA1function include compounds that antagonize an in vitro or in vivofunctional activity of TRPA1. When a particular functional activity isonly readily observable in an in vitro assay, the ability of a compoundto inhibit TRPA1 function in that in vitro assay serves as a reasonableproxy for the activity of that compound. In certain embodiments, aneffective amount is an amount sufficient to inhibit a TRPA1-mediatedcurrent and/or the amount sufficient to inhibit TRPA1 mediated ion flux.

The term “hydrate” as used herein, refers to a compound formed by theunion of water with the parent compound.

The term “preventing,” when used in relation to a condition, such as alocal recurrence (e.g., pain), a disease such as cancer, a syndromecomplex such as heart failure or any other medical condition, is wellunderstood in the art and includes administration of a composition whichreduces the frequency of, or delays the onset of, symptoms of a medicalcondition in a subject relative to a subject which does not receive thecomposition. Thus, prevention of cancer includes, for example, reducingthe number of detectable cancerous growths in a population of patientsreceiving a prophylactic treatment relative to an untreated controlpopulation, and/or delaying the appearance of detectable cancerousgrowths in a treated population versus an untreated control population,e.g., by a statistically and/or clinically significant amount.Prevention of an infection includes, for example, reducing the number ofdiagnoses of the infection in a treated population versus an untreatedcontrol population, and/or delaying the onset of symptoms of theinfection in a treated population versus an untreated controlpopulation. Prevention of pain includes, for example, reducing themagnitude of, or alternatively delaying, pain sensations experienced bysubjects in a treated population versus an untreated control population.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity in the host animal.

The term “solvate” as used herein, refers to a compound formed bysolvation (e.g., a compound formed by the combination of solventmolecules with molecules or ions of the solute).

The terms “TRPA1”, “TRPA1 protein”, and “TRPA1 channel” are usedinterchangeably throughout the application. These terms refer to an ionchannel (e.g., a polypeptide) comprising the amino acid sequence setforth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 of WO 2007/073505,or an equivalent polypeptide, or a functional bioactive fragmentthereof. In certain embodiments, the term refers to a polypeptidecomprising, consisting of, or consisting essentially of, the amino acidsequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. TRPA1includes polypeptides that retain a function of TRPA1 and comprise (i)all or a portion of the amino acid sequence set forth in SEQ ID NO: 1,SEQ ID NO: 3 or SEQ ID NO: 5; (ii) the amino acid sequence set forth inSEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 with 1 to about 2, 3, 5, 7,10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions;(iii) an amino acid sequence that is at least 70%, 75%, 80%, 90%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ IDNO: 5; and (iv) functional fragments thereof. Polypeptides of theinvention also include homologs, e.g., orthologs and paralogs, of SEQ IDNO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

The “enantiomeric excess” or “% enantiomeric excess” of a compositioncan be calculated using the equation shown below. In the example shownbelow a composition contains 90% of one enantiomer, e.g., the Senantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.

ee=(90−10)/100=80%.

Thus, a composition containing 90% of one enantiomer and 10% of theother enantiomer is said to have an enantiomeric excess of 80%.

The “diastereomeric excess” or “% diastereomeric excess” of acomposition can be calculated using the equation shown below. In theexample shown below a composition contains 90% of one diastereomer, and10% of another enantiomer.

de=(90−10)/100=80%.

Thus, a composition containing 90% of one diastereomer and 10% of theother diastereomer is said to have a diastereomeric excess of 80%.

Chemical Definitions

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

For compounds of the invention in which a variable appears more thanonce, each variable can be a different moiety selected from the Markushgroup defining the variable. For example, where a structure is describedhaving two R groups that are simultaneously present on the samecompound; the two R groups can represent different moieties selectedfrom the Markush group defined for R.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

As used herein, “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, and canhave a number of carbon atoms optionally designated (i.e., C₁-C₆ meansone to six carbons). Examples of saturated hydrocarbon groups include,but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl,homologs and isomers of, for example, n-pentyl, n-hexyl, and the like.

As used herein, “alkylene” refers to a divalent alkyl, e.g., —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, and—CH₂CH₂CH₂CH₂CH₂CH₂—.

As used herein, “alkenyl” can be a straight or branched hydrocarbonchain, containing at least one double bond, and having from two to sixcarbon atoms (i.e. C₂-C₆ alkenyl). Examples of alkenyl groups, include,but are not limited to, groups such as ethenyl (i.e., vinyl),prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl,and the like.

As used herein, “alkoxy” can be a straight chain or branched alkoxygroup having from one to six carbon atoms (i.e., C₁-C₆ alkoxy). Examplesof alkoxy groups, include, but are not limited to, groups such asmethoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy,tert-butyloxy, pentyloxy, or hexyloxy, and the like.

As used herein, “alkynyl” can be a straight or branched hydrocarbonchain, containing at least one triple bond, having from two to sixcarbon atoms (i.e. C₂-C₆ alkynyl). Examples of alkynyl groups, include,but are not limited to, groups such as ethynyl, propynyl, butynyl,pentynyl, hexynyl, and the like.

As used herein, “amino” or “amine” refers to a —NH₂ radical group.

As used herein, “aryl” refers to a polyunsaturated, aromatic,hydrocarbon moiety which can be a single ring or multiple rings (e.g., 1to 2 rings) which are fused together or linked covalently, having fromsix to twelve carbon atoms (i.e. C₆-C₁₂ aryl). Non-limiting examples ofaryl groups include phenyl, 1-naphthyl, 2-naphthyl, and 4-biphenyl.

As used herein, “cycloalkyl” refers to a monocyclic or polycyclicradical that contains only carbon and hydrogen, and may be saturated, orpartially unsaturated. Cycloalkyl groups include groups having from 3 to10 ring atoms (i.e. C₃-C₁₀ cycloalkyl). Examples of cycloalkyl groupsinclude, but are not limited to, groups such as cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloseptyl,cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.

As used herein, “halo” or “halogen,” independently or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. The term “halide” by itself or as part ofanother substituent, refers to a fluoride, chloride, bromide, or iodideatom.

As used herein, “haloalkyl” and “haloalkoxy” can include alkyl andalkoxy structures that are substituted with one or more halo groups orwith combinations thereof. For example, the terms “fluoroalkyl” and“fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, inwhich the halo is fluorine.

As used herein, “heteroalkyl” can include an optionally substitutedalkyl, which has one or more skeletal chain atoms selected from an atomother than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus orcombinations thereof. A numerical range may be given, e.g. C₁-C₆heteroalkyl which refers to the number of carbons in the chain, which inthis example includes 1 to 6 carbon atoms. For example, a —CH₂OCH₂CH₃radical is referred to as a “C₃” heteroalkyl. Connection to the rest ofthe molecule may be through either a heteroatom or a carbon in theheteroalkyl chain.

As used herein, “heteroaryl” refers to a 5- to 14-membered aromaticradical (e.g., C₂-C₁₃ heteroaryl) that includes one or more ringheteroatoms selected from nitrogen, oxygen and sulfur, and which may bea monocyclic or bicyclic ring system. Bivalent radicals derived fromunivalent heteroaryl radicals whose names end in “-yl” by removal of onehydrogen atom from the atom with the free valence are named by adding“-idene” to the name of the corresponding univalent radical, e.g., apyridyl group with two points of attachment is a pyridylidene. AnN-containing “heteroaromatic” or “heteroaryl” moiety refers to anaromatic group in which at least one of the skeletal atoms of the ringis a nitrogen atom. The polycyclic heteroaryl group may be fused ornon-fused. The heteroatom(s) in the heteroaryl radical is optionallyoxidized. One or more nitrogen atoms, if present, are optionallyquaternized. The heteroaryl is attached to the rest of the moleculethrough any atom of the ring(s). Examples of heteroaryl groups includewithout limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl, imidazolyl,thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl,benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl,1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl,benzimidazolyl, indolinyl, and the like.

As used herein, “heterocyclyl” or “heterocycloalkyl” can be a stable 3-to 18-membered non-aromatic mono, di, or tricyclic heterocycle ringradical that comprises two to twelve carbon atoms and from one to sixheteroatoms selected from nitrogen, oxygen and sulfur. Examples ofheterocycloalkyl groups include, but are not limited to, groups such asdioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, azetidinyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, and the like.

As used herein, “hydroxy” or “hydroxyl” refers to —OH.

As used herein, “cyano” refers to —CN.

As used herein, “nitro” refers to —NO₂.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds (e.g., alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, any of which mayitself be further substituted), as well as halogen, carbonyl (e.g.,aldehyde, ketone, ester, carboxyl, or formyl), thiocarbonyl (e.g.,thioester, thiocarboxylate, or thioformate), amino (e.g.,—N(R^(b))(R^(c)), wherein each R^(b) and R^(c) is independently H orC₁-C₆ alkyl), cyano, nitro, —SO₂N(R^(b))(R^(c)), —SOR^(d), and S(O)₂R″,wherein each R^(b), R^(c), and R^(d) is independently H or C₁-C₆ alkyl.Illustrative substituents include, for example, those described hereinabove. The permissible substituents can be one or more and the same ordifferent for appropriate organic compounds. For purposes of thisinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit TRPA1activity), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Compounds

Described herein are compounds, which can be useful in the treatment ofa disorder where inhibition of TRPA1 is beneficial. Such disorders aredescribed herein.

The compounds include compounds of Formula (I)

wherein:

R¹ is H, C₁-C₆ alkyl, C₁-C₆ alkenyl, or C₁-C₆ alkynyl;

R² is H, C₁-C₆ alkyl, C₁-C₆ alkenyl, or C₁-C₆ alkynyl optionallysubstituted with one or more R⁵ groups;

R³ is H, C₁-C₆ alkyl, C₁-C₆ alkenyl, or C₁-C₆ alkynyl;

R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino,dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido, thioyl,sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups;

R⁵ is independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl,hydroxy, amino, amido, phosphonate, carboxyl, ether, alkylthio,haloalkyl, and cyano; and

R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, alkehyde, ester, heterocycle, an aromatic orheteroaromatic ring, haloalkyl, and cyano.

In some embodiments, R¹ is C₁-C₆ alkyl, for example, —CH₃. In someembodiments, R¹ is H.

In some embodiments, R² is H or C₁-C₆ alkyl, for example, —CH₃, —CD₃, or—CHF₂.

In some embodiments, each R¹ and R² is independently C₁-C₆ alkyl, forexample, —CH₃. In some embodiments, each R¹ and R² is independently —CH₃and R³ is H.

In some embodiments, R³ is H. In some embodiments, R³ is C₁-C₆ alkyl,for example, —CH₃.

In some embodiments, each of R¹ and R² and R³ is independently C₁-C₆alkyl, for example, —CH₃.

In some embodiments, the compound of the Formula (I) is the compound ofthe Formula (Ia):

In some embodiments, each of R¹ and R² and R³ is independently C₁-C₆alkyl, for example, —CH₃.

In some embodiments, the compound of the Formula (I) of claim 1, is thecompound of the Formula (Ib):

In some embodiments, each of R¹ and R² and R³ is independently C₁-C₆alkyl, for example, —CH₃.

In some embodiments, R⁴ is heterocyclyl, for example, a 4 to 8-memberedring. In some embodiments, the heterocyclyl is linked through a nitrogenatom. In some embodiments, R⁴ is substituted heterocyclyl. In someembodiments, R⁴ is selected from the group:

In some embodiments, R⁴ is selected from the group:

and m is 1.

In some embodiments, R⁴ is selected from the group:

In some embodiments, R⁴ is selected from the group:

and m is 1.

In some embodiments, R⁴ is selected from the group:

In some embodiments, m is 0. In some embodiments, m is 1.

In some embodiments, R⁶ is, alkyl, haloalkyl, or cyano, for example,alkyl or haloalkyl, such as —CF₃.

In some embodiments, R⁴ is selected from the group:

In some embodiments, the compound of Formula (I) is of the Formula (II):

wherein:

n is an integer from 0 to 4; and

m is selected from an integer from 0 to 4.

In some embodiments, the compound of Formula (I) is of the Formula(IIa):

wherein:

n is an integer from 0 to 4; and

m is selected from an integer from 0 to 4.

In some embodiments, the compound of Formula (I) is of the Formula(IIb):

wherein:

n is an integer from 0 to 4; and

m is selected from an integer from 0 to 4.

In some embodiments, the compound is selected from the following group:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the following group:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of Formula (I) has a melting pointgreater than or equal to about 100° C. In some embodiments, saidcompound of Formula (I) has a melting point greater than or equal toabout 125° C., about 150° C., about 175° C., or about 180° C. In someembodiments, said compound of Formula (I) has a melting point in therange of about 180° C. to about 205° C. In some embodiments, saidcompound of Formula (I) has a melting point in the range of about 190°C. to about 200° C. In some embodiments, said compound of Formula (I)has a melting point in the range of about 190° C. to about 196° C.

In some embodiments, a solid crystalline form of a compound of Formula(I) is produced upon slurry treatment with a suitable solvent (e.g.,ethanol, water, or a combination thereof). In some embodiments, a solidcrystalline form (e.g., an anhydrous solid crystalline form) of acompound of Formula (I) is produced upon slurry treatment with asuitable solvent (e.g., ethanol, water, or a combination thereof)followed by an additional treatment (e.g., vacuum treatment, e.g., −80°C. for one day).

In some embodiments, a solid crystalline form of a compound of Formula(I) (e.g., produced upon slurry treatment with a suitable solvent, e.g.,ethanol, water, or a combination thereof, and optionally followed by anadditional treatment, e.g., vacuum treatment, e.g., −80° C. for one day)has a melting point greater than or equal to about 100° C. In someembodiments, said solid crystalline form of a compound of Formula (I)has a melting point greater than or equal to about 125° C., about 150°C., about 175° C., or about 180° C. In some embodiments, said solidcrystalline form of a compound of Formula (I) has a melting point in therange of about 180° C. to about 205° C. In some embodiments, said solidcrystalline form of a compound of Formula (I) has a melting point in therange of about 190° C. to about 200° C. In some embodiments, said solidcrystalline form of a compound of Formula (I) has a melting point in therange of about 190° C. to about 196° C.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof, which is referred to asCompound 2, Example 2, or Compound of Example 2 herein.

In some embodiments, a solid crystalline form of Compound 2 (e.g., FormA) is produced upon slurry treatment with a suitable solvent (e.g.,ethanol, water, or a combination thereof). In some embodiments, saidsolid crystalline form of Compound 2 (e.g., Form A) has an X-ray powderdiffraction pattern comprising characteristic peaks, expressed in termsof 2θ, at one or more of the following angles: about 7.67°, about12.52°, about 13.49°, and about 19.31°. In some embodiments, said solidcrystalline form of Compound 2 (e.g., Form A) has characteristic peaksas shown in FIG. 1.

In some embodiments, a solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) isproduced upon slurry treatment with a suitable solvent (e.g., ethanol,water, or a combination thereof) followed by an additional treatment(e.g., vacuum treatment, e.g., −80° C. for one day). In someembodiments, said solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) has anX-ray powder diffraction pattern comprising characteristic peaks,expressed in terms of 2θ, at one or more of the following angles: about9.78°, about 12.98°, about 19.20°, and about 19.67°. In someembodiments, said solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) hascharacteristic peaks as shown in FIG. 2.

In some embodiments, a solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) has amelting point greater than or equal to about 100° C. In someembodiments, said solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) has amelting point greater than or equal to about 125° C., about 150° C.,about 175° C., or about 180° C. In some embodiments, said solidcrystalline form of Compound 2 (e.g., an anhydrous solid crystallineform of Compound 2, e.g., Form B) has a melting point in the range ofabout 180° C. to about 205° C. In some embodiments, said solidcrystalline form of Compound 2 (e.g., an anhydrous solid crystallineform of Compound 2, e.g., Form B) has a melting point in the range ofabout 190° C. to about 200° C. In some embodiments, said solidcrystalline form of Compound 2 (e.g., an anhydrous solid crystallineform of Compound 2, e.g., Form B) has a melting point in the range ofabout 190° C. to about 196° C. In some embodiments, said solidcrystalline form of Compound 2 (e.g., an anhydrous solid crystallineform of Compound 2, e.g., Form B) has a differential scanningcalorimetry trace as shown in FIG. 3.

In some embodiments, a solid crystalline form of Compound 2 (e.g., ananhydrous solid crystalline form of Compound 2, e.g., Form B) isproduced upon slurry treatment with a suitable solvent (e.g., ethanol,water, or a combination thereof) followed by an additional treatment(e.g., vacuum treatment, e.g., −80° C. for one day) wherein said solidcrystalline form of Compound 2 (e.g., an anhydrous solid crystallineform of Compound 2, e.g., Form B) has a melting point greater than orequal to about 150° C. and an X-ray powder diffraction patterncomprising characteristic peaks, expressed in terms of 2θ, at one ormore of the following angles: about 9.78°, about 12.98°, about 19.20°,and about 19.67°. In some embodiments, a solid crystalline form ofCompound 2 (e.g., an anhydrous solid crystalline form of Compound 2,e.g., Form B) is produced upon slurry treatment with a suitable solvent(e.g., ethanol, water, or a combination thereof) followed by anadditional treatment (e.g., vacuum treatment, e.g., −80° C. for one day)wherein said solid crystalline form of Compound 2 (e.g., an anhydroussolid crystalline form of Compound 2, e.g., Form B) has a melting pointin the range of 185° C. to about 205° C. and an X-ray powder diffractionpattern comprising characteristic peaks, expressed in terms of 2θ, atone or more of the following angles: about 9.78°, about 12.98°, about19.20°, and about 19.67°.

Certain embodiments of the present invention comprise a purifiedpharmaceutical preparation comprising a compound of Formula (I). In someembodiments, the pharmaceutical preparation comprises a diastereomericexcess of greater than or equal to about 55% (e.g., about 60%, about70%, about 80%, about 90%, about 95%, about 99%, or about 99.5%) of onediastereomer over another diastereomer. In some embodiments, thepharmaceutical preparation comprises a diastereomeric excess of greaterthan or equal to about 95% of one diastereomer over anotherdiastereomer. In some embodiments, the pharmaceutical preparationcomprises a diastereomeric excess of greater than or equal to about 99%of one diastereomer over another diastereomer.

In some embodiments, the pharmaceutical preparation comprises less thanor equal to about 10% moisture content (e.g., water content). In someembodiments, the pharmaceutical composition comprises less than or equalto about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%,about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, orabout 0.001% moisture content (e.g., water content). In someembodiments, the pharmaceutical preparation is substantially free ofmoisture (e.g., water).

In some embodiments, the pharmaceutical preparation comprises a compoundof Formula (I), wherein the compound is:

or a pharmaceutically acceptable salt thereof, which is referred to asCompound 2, Example 2, or Compound of Example 2 herein.

In some embodiments, the pharmaceutical preparation comprises a compoundof Formula (I), wherein the compound is Compound 2, or apharmaceutically acceptable salt thereof, and the preparation has adiastereomeric excess of Compound 2 greater than or equal to about 99%.In some embodiments, the pharmaceutical preparation comprises a compoundof Formula (I), wherein the compound is Compound 2, or apharmaceutically acceptable salt thereof, and the preparation has amoisture content (e.g., water content) of less than or equal to about0.1%. In some embodiments, the pharmaceutical preparation comprises acompound of Formula (I), wherein the compound is Compound 2, or apharmaceutically acceptable salt thereof, and the preparation has adiastereomeric excess of Compound 2 greater than or equal to about 99%and a moisture content (e.g., water content) of less than or equal toabout 0.1%.

In some embodiments, the pharmaceutical preparation comprises a solidcrystalline form of Compound 2 (e.g., Form A) that has an X-ray powderdiffraction pattern comprising characteristic peaks, expressed in termsof 2θ, at one or more of the following angles: about 7.67°, about12.52°, about 13.49°, and about 19.31°, and the preparation has adiastereomeric excess of Compound 2 greater than or equal to about 99%and a moisture content (e.g., water content) of less than or equal toabout 0.1%.

In some embodiments, the pharmaceutical preparation comprises a solidcrystalline form of Compound 2 (e.g., Form B) that has a melting pointin the range of 185° C. to about 205° C. and an X-ray powder diffractionpattern comprising characteristic peaks, expressed in terms of 2θ, atone or more of the following angles: about 9.78°, about 12.98°, about19.20°, and about 19.67°, and the preparation has a diastereomericexcess of Compound 2 greater than or equal to about 99% and a moisturecontent (e.g., water content) of less than or equal to about 0.1%.

Compounds of Formula (I) include molecules having an aqueous solubilitysuitable for oral or parenteral (e.g., intravenous) administrationleading to or resulting in the treatment of a disorder described herein,for example the treatment of pain. In some embodiments, the compound isformulated into a composition suitable for oral administration. Thepotency in inhibiting the TRPA1 ion channel of compounds of Formula (I)described herein was measured using the method of Example 33. Table 14discloses the TRPA1 inhibition in vitro potency of exemplary compounds(measured by the method of Example 33).

Preferred compounds of Formula (I) include compounds that inhibit theTRPA1 ion channel with a IC₅₀ value obtained by the method of Example 33of less than about 100 nM (preferably, less than about 75 nM, morepreferably less than about 25 nM).

Compounds of Formula (I) can inhibit the TRPA1 ion channel. In someembodiments, a compound of Formula (I) can be administered as part of anoral or parenteral (e.g., intravenous) pharmaceutical composition totreat a disorder described herein (e.g., pain) in a therapeuticallyeffective manner.

Certain compounds disclosed herein may exist in particular geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and trans-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof, as falling within the scope of theinvention. For example, if one chiral center is present in a molecule,the invention includes racemic mixtures, enantiomerically enrichedmixtures, and substantially enantiomerically or diastereomerically purecompounds. The composition can contain, e.g., more than 50%, more than60%, more than 70%, more than 80%, more than 90%, more than 95%, or morethan 99% of a single enantiomer or diastereomer. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

The compounds described herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compoundsdisclosed herein, whether radioactive or not, are intended to beencompassed within the scope of the present invention. For example,deuterated compounds and compounds incorporated ¹³C are intended to beencompassed within the scope of the invention.

Certain compounds disclosed herein can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Certain compounds disclosed herein mayexist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by the presentinvention and are intended to be within the scope of the presentinvention.

Pharmaceutical Compositions

Pharmaceutical compositions containing compounds described herein suchas a compound of Formula (I) or pharmaceutically acceptable salt thereofcan be used to treat or ameliorate a disorder described herein, forexample, a disorder responsive to the inhibition of the TRPA1 ionchannel in subjects (e.g., humans and animals).

The amount and concentration of compounds of Formula (I) in thepharmaceutical compositions, as well as the quantity of thepharmaceutical composition administered to a subject, can be selectedbased on clinically relevant factors, such as medically relevantcharacteristics of the subject (e.g., age, weight, gender, other medicalconditions, and the like), the solubility of compounds in thepharmaceutical compositions, the potency and activity of the compounds,and the manner of administration of the pharmaceutical compositions. Forfurther information on Routes of Administration and Dosage Regimes thereader is referred to Chapter 25.3 in Volume 5 of ComprehensiveMedicinal Chemistry (Corwin Hansch; Chairman of Editorial Board),Pergamon Press 1990.

While it is possible for a compound disclosed herein to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation, where the compound is combined with one or morepharmaceutically acceptable excipients or carriers. The compoundsdisclosed herein may be formulated for administration in any convenientway for use in human or veterinary medicine. In certain embodiments, thecompound included in the pharmaceutical preparation may be activeitself, or may be a prodrug, e.g., capable of being converted to anactive compound in a physiological setting.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable carriers include: (1) sugars,such as lactose, glucose and sucrose; (2) starches, such as corn starchand potato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21)cyclodextrins such as Captisol®; and (22) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Solid dosage forms (e.g., capsules, tablets, pills, dragees, powders,granules and the like) can include one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents.

Liquid dosage forms can include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Ointments, pastes, creams and gels may contain, in addition to an activecompound, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions disclosed herein, such as dragees, capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The formulations disclosed herein can be delivered via a device.Exemplary devices include, but are not limited to, a catheter, wire,stent, or other intraluminal device. Further exemplary delivery devicesalso include a patch, bandage, mouthguard, or dental apparatus.Transdermal patches have the added advantage of providing controlleddelivery of a compound disclosed herein to the body. Such dosage formscan be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, drops, solutions and the like,are also contemplated as being within the scope of this invention.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds disclosed herein are administered as pharmaceuticals,to humans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1 to 99.5% (more preferably, 0.5to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

The formulations can be administered topically, orally, transdermally,rectally, vaginally, parenterally, intranasally, intrapulmonary,intraocularly, intravenously, intramuscularly, intraarterially,intrathecally, intracapsularly, intraorbitally, intracardiacly,intradermally, intraperitoneally, transtracheally, subcutaneously,subcuticularly, intraarticularly, subcapsularly, subarachnoidly,intraspinally, intrasternally or by inhalation.

One specific embodiment is an antitussive composition for peroraladministration comprising an agent that inhibits both a TRPA1-mediatedcurrent with an IC₅₀ of 1 micromolar or less, and an orally-acceptablepharmaceutical carrier in the form of an aqueous-based liquid, or soliddissolvable in the mouth, selected from the group consisting of syrup,elixer, suspension, spray, lozenge, chewable lozenge, powder, andchewable tablet. Such antitussive compositions can include one or moreadditional agents for treating cough, allergy or asthma symptom selectedfrom the group consisting of: antihistamines, 5-lipoxygenase inhibitors,leukotriene inhibitors, H3 inhibitors, β-adrenergic receptor agonists,xanthine derivatives, α-adrenergic receptor agonists, mast cellstabilizers, expectorants, and NK1, NK2 and NK3 tachykinin receptorantagonists.

Still another embodiment is a metered dose aerosol dispenser containingan aerosol pharmaceutical composition for pulmonary or nasal deliverycomprising an agent that inhibits a TRPA1-mediated current with an IC₅₀of 1 micromolar or less. For instance, it can be a metered dose inhaler,a dry powder inhaler or an air-jet nebulizer.

Dosages

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound disclosed hereinemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular, intrathecal and subcutaneous doses of thecompounds described herein for a subject will range from about 0.0001 toabout 100 mg per kilogram of body weight per day. For example, the dosecan be 1-50, 1-25, or 5-10 mg/kg. Generally, oral doses of the compoundsdescribed herein for a subject will range from about 1 to about 1,000mg/day (e.g., from about 5 to about 500 mg/day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Methods of Treatment

The compounds described herein can be used to treat or prevent adisorder described herein. For example, compounds with TRPA1 inhibitoryactivity are provided herein for the prevention, treatment, oralleviating symptoms of a disease or condition associated with TRPALCompounds of Formula (I), or pharmaceutical compositions containing oneor more compounds of Formula (I), can be administered to treatdisorders, conditions, or diseases described herein such as thosetreatable by the inhibition of TRPAL For example, the pharmaceuticalcompositions comprising compounds of Formula (I), or pharmaceuticallyacceptable salts thereof, are useful as a perioperative analgesic, forexample in the management of mild to moderate acute post-operative painand management of moderate to severe acute pain as an adjunct to opioidanalgesics. The pharmaceutical compositions comprising atherapeutically-effective dose of compounds of Formula (I), can beadministered to a patient for treatment of pain in a clinically safe andeffective manner, including one or more separate administrations of thepharmaceutical compositions comprising compounds of Formula (I).Additional exemplary methods include the treatment of peripheraldiabetic neuropathy (PDN) and chemotherapy induced peripheral neuropathy(CIPN). For example, a pharmaceutical composition comprising atherapeutically effective dose of compounds of Formula (I), orpharmaceutically acceptable salts thereof can be administered (e.g.,intravenously) to a subject in need thereof multiple times per day(e.g., BID) over a course of treatment of one or more days to treat painin the subject. Pharmaceutical compositions comprising compounds ofFormula (I) can also be used to treat or ameliorate pulmonaryconditions, such as obstructive diseases, e.g., chronic obstructivepulmonary disease (COPD), asthma (e.g., cold induced asthma,exercise-induced asthma, allergy-induced asthma, and occupationalasthma), and cough.

Those of skill in the treatment of diseases linked to the mediation ofthe TRPA1 receptor will be able to determine the therapeuticallyeffective amount of a compound of Formula (I) from the test resultspresented hereinafter. In general, a suitable daily dose of a compoundof the invention will be that amount of the compound that is the lowestdose able to produce a therapeutic effect. Such an effective dose willgenerally depend upon various factors. Generally, oral, sublingual,rectal, intravenous, topical, transdermal, inhaled andintracerebroventricular doses of the compounds of this invention for apatient will range from about 0.0001 to about 100 mg per kilogram ofbody weight per day. For example, the dose can be 1-50, 1-25, or 5-10mg/kg. It is contemplated, for instance, that a therapeuticallyeffective dose will be from about 0.001 mg/kg to about 50 mg/kg per kgof body weight, more preferably from about 0.01 mg/kg to about 10 mg/kgper kg of body weight of the patient to be treated. It may beappropriate to administer the therapeutically effective dose in the formof two or more sub-doses at appropriate intervals throughout the day.Said sub-doses may be formulated as unit dosage forms, for example eachcontaining from about 0.1 mg to about 1000 mg, more particularly fromabout 1 to about 500 mg, of the active ingredient per unit dosage form.

The exact dosage and frequency of administration depends on theparticular compound of formula (I) used, the particular condition beingtreated, the severity of the condition being treated, the age, weightand general physical condition of the particular patient as well as theother medication the patient may be taking, as is well known to thoseskilled in the art. Furthermore, said “therapeutically effective amount”may be lowered or increased depending on the response of the treatedpatient and/or depending on the evaluation of the physician prescribingthe compounds of the instant invention. The effective daily amountranges mentioned hereinabove are therefore only guidelines. A physicianor veterinarian having ordinary skill in the art can readily determineand prescribe the effective amount of the pharmaceutical compositionrequired.

Exemplary disorders suitable for treatment with a compound orcomposition described herein are provided below.

Pain

The compounds of Formula (I) that are useful in the modulation of TRPA1can be used in the formulation of analgesic pharmaceuticals suitable forthe treatment and/or prophylaxis of pain in mammals, especially inhumans. Endogenous activators of TRPA1 are produced during manypathological conditions including tissue injury, inflammation, andmetabolic stress. Compounds and pharmaceutical compositions of thepresent invention can be administered to treat pain resulting fromactivation of TRPA1 including neuropathic pain. Relevant neuropathicpain conditions include, but are not limited to, painful diabeticneuropathy, chemotherapy-induced peripheral neuropathy, lower back pain,trigeminal neuralgia, post-herpetic neuralgia, sciatica, and complexregional pain syndrome

Compositions and methods provided herein may also be used in connectionwith treatment of in the treatment of inflammation and inflammatorypain. Such disorders include rheumatoid arthritis, osteoarthritis,temperomandibular disorder. In some embodiments, the compositions andmethods provided herein may be used to treat headache pain, e.g.,migraine.

Disclosed compounds also may be useful in the treatment of visceral painand inflammation. Relevant diseases include pancreatitis, inflammatorybowel disease, colitis, Crohn's disease, endometriosis, pelvic pain, andangina.

Additional exemplary pain indications for which compounds disclosedherein can be used include temperomandibular disorder, cancer pain(resulting either from the underlying disease or from the treatments),burn pain, oral pain, oral pain due to cancer treatment, crush andinjury induced pain, incisional pain, bone pain, sickle cell diseasepain, fibromyalgia and musculoskeletal pain. TRPA1 has been show to playa role in cancer related pain (see, e.g., Trevisan et al., Cancer Res(2013) 73(10):3120-3131); postoperative pain (see, e.g., Wei et al,Anasthesiology (2012) 117:137-148); pathological pain (see, e.g., Chenet al, Pain (2011) 152:2549-2556); and pain related to chemical injury(see, e.g., Macpherson et al, J Neurosci (2007) 27(42):11412-11415).

Hyperalgesia (e.g., mechanical hyperalegsia, cold hyperalegsia) orincreased sensitivity to pain (e.g., acute, chronic). Multiple ChemicalSensitivity is a disorder linked to chemical exposure with multi-organsymptoms including respiratory symptoms and headache.

Allodynia (e.g., cutaneous allodynia, e.g., cephalic, extracephalic) isa pain due to a stimulus which does not normally provoke pain, e.g.,temperature or physical stimuli, and differs from hyperalgesia, whichgenerally refers to an extreme, exaggerated reaction to a stimulus whichis normally painful.

Migraine

The compounds of Formula (I) that are useful in the modulation of TRPA1can be used in the formulation of pharmaceuticals suitable for thetreatment and/or prophylaxis of migraine in mammals, especially inhumans. Exposure to TRPA1 activators has been shown to trigger migrainein susceptible populations. Such activators include but are not limitedto umbellulone, nitroglycerin, cigarette smoke, and formaldehyde.Accordingly, TRPA1 antagonists of the invention represent a significantpossible therapeutic for the treatment of both chronic and acutemigraine.

Inflammatory Diseases and Disorders

Compositions and methods provided herein may also be used in connectionwith treatment of inflammatory diseases. These diseases include but arenot limited to asthma, chronic obstructive pulmonary disease, rheumatoidarthritis, osteoarthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases such as multiplesclerosis, and disorders of the immune system. TRPA1 has been show toplay a role in pancreatic pain and inflammation (see, e.g., Schwartz etal., Gastroenterology (2011) 140(4):1283-1291).

Peripheral neuropathy, for example diabetic neuropathy (e.g., painfuldiabetic neuropathy), is a particular condition that involves both aneuronal and an inflammatory component. Without being bound by amechanistic theory, the TRPA1 antagonists of the invention may be usefulin treating peripheral neuropathies including, but not limited to,diabetic neuropathy. In addition to their use in the treatment ofperipheral neuropathies (e.g., reducing inflammation), the subjectinhibitors may also be useful in reducing the pain associated withperipheral neuropathy. TRPA1 has been show to play a role in neuropathyand neuropathic pain (see, e.g., Wei et al, Anesthesiology (2009)111:147-54; Koivisto et al., Pharmacol Res (2011) 65:149-158).

Neurogenic inflammation often occurs when neuronal hyperexcitabilityleads to the release of peptides that trigger inflammation. Thesepeptides include substance P and CGRP. Blocking TRPA1 would reduceneuronal activity and thus could block neurogenic inflammation. Forexample, neurogenic inflammation in the respiratory tract, can result inasthma and allergic rhinitis symptoms, and neurogenic inflammation inthe dura may also mediate migraine pain.

Pancreatitis

Pancreatitis is an inflammation of the pancreas. The pancreas is a largegland behind the stomach and close to the duodenum. Normally, digestiveenzymes do not become active until they reach the small intestine, wherethey begin digesting food. But if these enzymes become active inside thepancreas, they start “digesting” the pancreas itself. TRPA1 has beenshow to play a role in pancreatic pain and inflammation (see, e.g.,Schwartz et al., Gastroenterology (2011) 140(4):1283-1291.).

Acute pancreatitis is usually, although not exclusively, caused bygallstones or by alcohol abuse. Acute pancreatitis usually begins withpain in the upper abdomen that may last for a few days. The pain may besevere and may become constant. The pain may be isolated to the abdomenor it may reach to the back and other areas. Sometimes, and for somepatients, the pain is sudden and intense. Other times, or for otherpatients, the pain begins as a mild pain that worsens after eating.Someone with acute pancreatitis often looks and feels very sick. Othersymptoms may include swollen and tender abdomen, nausea, vomiting,fever, and rapid pulse. Severe cases of acute pancreatitis may causedehydration and low blood pressure, and may even lead to organ failure,internal bleeding, or death.

During acute pancreatitis attacks, the blood levels of amylase andlipase are often increased by at least 3-fold. Changes may also occur inblood levels of glucose, calcium, magnesium, sodium, potassium, andbicarbonate.

The current treatment depends on the severity of the attack. Treatment,in general, is designed to support vital bodily functions, manage pain,and prevent complications. Although acute pancreatitis typicallyresolved in a few days, pain management during an attack is oftenrequired. The compounds disclosed herein can be used to relieve the painassociated with acute pancreatitis.

Chronic pancreatitis may develop if injury to the pancreas continues.Chronic pancreatitis occurs when digestive enzymes attack and destroythe pancreas and nearby tissues, causing scarring and pain. Chronicpancreatitis may be caused by alcoholism, or by blocked, damaged, ornarrowed pancreatic ducts. Additionally, hereditary factors appear toinfluence the disease, and in certain cases, there is no identifiablecause (so called idiopathic pancreatitis).

Most people with chronic pancreatitis have abdominal pain. The pain mayget worse when eating or drinking, spread to the back, or becomeconstant and disabling. Other symptoms include nausea, vomiting, weightloss, and fatty stools.

Relieving pain is the first step in treating chronic pancreatitis. Oncethe pain has been managed, a high carbohydrate and low fat dietary planis put in place. Pancreatic enzymes may be used to help compensate fordecrease enzyme production from the injured pancreas. Sometimes insulinor other drugs are needed to control blood glucose.

Although pain is typically managed using drug therapy, surgery may benecessary to relieve pain. Surgery may be necessary to drain an enlargedpancreatic duct or even to 10 removing a portion of a seriously injuredpancreas.

Pain is frequently present with chronic pancreatitis. For example, painis present for approximately 75% of patients with alcoholic chronicpancreatitis, 50% of patients with lateonset idiopathic chronicpancreatitis, and 100% of patients with early-onset idiopathic chronicpancreatitis (DiMagno, Gastroenterology (1999) 116(5):1252-1257).

A minority of patients with pain have readily identifiable lesions whichare relatively easy to treat surgically or endoscopically. In otherpatients, pain is often thought to result from a variety of causes,including elevated intrapancreatic pressure, ischemia, and fibrosis.Without being bound by theory, however, these phenomena are not likelythe underlying cause of the pain. Rather, pain may result from abackground of neuronal sensitization induced by damage to theperineurium and subsequent exposure of the nerves to mediators andproducts of inflammation.

Given the importance of effective pain management in patients withchronic pancreatitis, additional therapies for treating painful symptomsare important and useful. The compounds disclosed herein can be used tomanage the pain associated with chronic pancreatitis; they can be usedalone or as part of an overall therapeutic treatment plan to managepatients with chronic pancreatitis. For example, the compounds can beadministered with pancreatic enzymes and/or insulin as part of atherapeutic regimen designed to manage patients with chronicpancreatitis.

Cancer treatments are not only painful, but they may even be toxic tohealthy tissue. Some chemotherapeutic agents can cause painfulneuropathy. Accordingly, the compounds disclosed herein could representa significant possible therapeutic for the treatment of the pain and/orinflammation associated with cancer treatments that cause neuropathy.

A major function of prostaglandins is to protect the gastric mucosa.Included in this function is the modulation of intracellular calciumlevel in human gastric cells which plays a critical role in cellproliferation. Consequently, inhibition of prostaglandins bynonsteroidal anti-inflammatory drugs (NSAIDs) can inhibit calcium influxin gastric cells (Kokoska et al. (1998) Surgery (St Louis)124(2):429-437). The NSAIDs that relieve inflammation most effectivelyalso produce the greatest gastrointestinal damage (Canadian FamilyPhysician, 5 Jan. 1998, p. 101). Thus, the ability to independentlymodulate calcium channels in specific cell types may help to alleviatesuch side effect of anti-inflammatory therapy. Additionally oralternatively, administration of TRPA1 inhibitory compounds disclosedherein may be used in combination with NSAIDs, thus promoting painrelief using reduced dosage of NSAIDs.

TRPA1 may mediate ongoing nociception in chronic pancreatitis; and maybe involved in transforming acute into chronic inflammation andhyperalgesia in pancreatitis. TRPA1 may also mediate irritation andburning in the e.g., nasal and oral mucosa and respiratory lining.

Neuropathy

Because TRPA1 overactivity can lead to a toxic calcium overload, TRPA1antagonists also have utility in the prevention of neuropathy associatedwith diabetes, chemical injury, chemotherapy, medicines such as statins,HIV/AIDS, Fabry's disease, vitamin deficiency, inherited polyneuropathysuch as Marie-Charcot Tooth disease, and trauma. Peripheralneurodegenerative diseases such as Amyotrophic Lateral Sclerosis mayalso be amenable to treatment with a TRPA1 antagonist.

Pulmonary Disease and Cough

Compositions and methods provided herein may also be used in connectionwith the treatment of pulmonary diseases, including, but not limited to,asthma (including exercise-induced asthma, atopic asthma, allergicasthma), Chronic Obstructive Pulmonary disease (COPD), emphysema, cysticfibrosis, bronchiectasis, bronchiolitis, allergic bronchopulmonaryaspergillosis, bronchiolitis obliterans (popcorn worker lung), diseasesdue to chemical exposure including exposures to diacetyl, formaldehyde,and other irritants. These conditions also include tuberculosis,restrictive lung disease including asbestosis, radiation fibrosis,hypersensitivity pneumonitis, infant respiratory distress syndrome,idiopathic pulmonary fibrosis, idiopathic interstial pneumoniasarcoidosis, eosinophilic pneumonia, lymphangioleiomyomatosis, pulmonaryLangerhan's cell histiocytosis, and pulmonary alveolar proteinosis;respiratory tract infections including upper respiratory tractinfections (e.g., common cold, sinusitis, tonsillitis, pharyngitis andlaryngitis) and lower respiratory tract infections (e.g., pneumonia);respiratory tumors whether malignant (e.g., small cell lung cancer,non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma,large cell undifferentiated carcinoma, carcinoid, mesothelioma,metastatic cancer of the lung, metastatic germ cell cancer, metastaticrenal cell carcinoma) or benign (e.g., pulmonary hamartoma, congenitalmalformations such as pulmonary sequestration and congenital cysticadenomatoid malformation (CCAM)); pleural cavity diseases (e.g., empyemaand mesothelioma); and pulmonary vascular diseases, e.g, pulmonaryembolism such as thromboembolism, and air embolism (iatrogenic),pulmonary arterial hypertension, pulmonary edema, pulmonary hemorrhage,inflammation and damage to capillaries in the lung resulting in bloodleaking into the alveoli. Other conditions that may be treated includedisorders that affect breathing mechanics (e.g., obstructive sleepapnea, central sleep apnea, Guillan-Barre syndrome, and myastheniagravis).

The present compounds can also be useful for treating, reducing, orpreventing cough (with or without the production of sputum), coughassociated with asthma, cough associated with influenza, coughing blood(haemoptysis), cough of unknown etiology, allergy-induced cough, andcough due to chemical exposures.

Dermatological Disorders

A number of agents that cause itch activate TRPA1 directly or viaactivation of receptors which couple to TRPA1 downstream. Compositionsand methods provided herein may also be used in connection with thetreatment of itch. Indications include, but are not limited to,conditions triggered by exposure to exogenous chemicals such as contactdermatitis, poison ivy, itch due to cancer including lymphomas, itchcaused by medications such as chloroquine, itch due to reactive drugmetabolites or itch due to dry skin.

Additional exemplary indications include atopic dermatitis, psoriasis,hives, eczema, dyshidrotic eczema, mouth ulcers, diaper rash.

Itch

Itch, or acute pruritus, while serving an important protective functionby e.g., warning against harmful agents in the environment, can also bea debilitating condition that e.g., accompanies numerous skin, systemicand nervous system disorders. Some forms of itch are mediated byhistamine signaling as such are susceptible to treatment with e.g.,antihistamines. However, most pathophysiological itch conditions areinsensitive to antihistamine treatment. Compounds and pharmaceuticalcompositions of the present invention can be administered to treat itch.

Atopic dermatitis (AD) is a chronic itch and inflammatory disorder ofthe skin. Patients with severe AD can develop asthma and allergicrhinitis, also known as atopic march. Skin rash and pruritus may beassociated with atopic disease. Chronic itch, e.g., in AD and psoriasis;includes pathophysiological hallmarks such as robust scratching,extensive epidermal hyperplasia from e.g., eczema, kidney failure,cirrhosis, nervous system disorders, some cancers.

Allergic contact dermatitis is a common skin disease associated withinflammation and persistent pruritus.

Methods as disclosed herein may inhibit skin edema, keratinocytehyperplasia, nerve growth, leukocyte infiltration, andantihistamine-resistant scratching behavior. Methods as disclosed hereinmay inhibit allergic response to e.g., exogenous stimulants, e.g.,haptens, oxazolone, urushiol (e.g., from poison ivy).

Disease and Injury Models

Compounds that antagonize TRPA1 function may be useful in theprophylaxis and treatment of any of the foregoing injuries, diseases,disorders, or conditions. In addition to in vitro assays of the activityof these compounds, their efficacy can be readily tested in one or moreanimal models. There are numerous animal models for studying pain. Thevarious models use various agents or procedures to simulate painresulting from injuries, diseases, or other condition (see, e.g.,Blackburn-Munro (2004) Trends in Pharmacol Sci (2004) 25:299-305 (e.g.,Tables 1, 3, or 4). Behavioral characteristics of challenged animals canthen be observed. Compounds or procedures that may reduce pain in theanimals can be readily tested by observing behavioral characteristics ofchallenged animals in the presence versus the absence of the testcompound(s) or procedure.

Exemplary behavioral tests used to study chronic pain include tests ofspontaneous pain, allodynia, and hyperalgesia. Id. To assess spontaneouspain, posture, gait, nocifensive signs (e.g., paw licking, excessivegrooming, excessive exploratory behavior, guarding of the injured bodypart, and self-mutilation) can be observed. To measure evoked pain,behavioral responses can be examined following exposure to heat (e.g.,thermal injury model).

Exemplary animal models of pain include, but are not limited to, themodels described in the Trevisan model, and the Koivisto referencesincluding streptozotocin induced painful diabetic neuropathy, bortexomibinduced peripheral neuropathy and oxaliplatin induced peripheralneuropathy; the Chung model, the spared nerve injury model, thecarageenan induced hyperalgesia model, the Freund's complete adjuvantinduced hyperalgesia model, the thermal injury model, the formalin modeland the Bennett Model.

In the Trevisan reference, chemotherapy-induced peripheral neuropathymodel involves the induction if a CIPN phenotype in mice by treatmentwith bortexomib or oxaliplatin (Trevisan et al, Cancer Res (2013) 73:3120-3131). Treatment of an animal with an inhibitor of TRPA1 can beevaluated using any of a variety of nociceptive tests such as the VonFrey hair test, the hot plate test, cold simulation, chemicalhyperalgesia, or the rotarod test.

The model of peripheral diabetic neuropathy (PDN) in the Koivistoreference involves induction of diabetes mellitus (DM) in rats withstreptozotocin, and assessing axon reflex induced by intraplantarinjection of a TRPA1 agonist (Koivisto et al., Phannacol Res (2011)65:149-158). Treatment with a compound that inhibits TRPA1 can beevaluated for the reduction in DM-induced attenuation of the cutaneousaxon reflex.

The Chung model of neuropathic pain (without inflammation) involvesligating one or more spinal nerves (see, e.g., Chung et al. Methods MolMed (2004) 99: 35-45; Kim and Chung, Pain (1992) 50: 355-363). Ligationof the spinal nerves results in a variety of behavioral changes in theanimals including heat hyperalgesia, cold allodynia, and ongoing pain.Compounds that antagonize TRPA1 can be administered to ligated animalsto assess whether they diminish these ligation-induced behavioralchanges in comparison to that observed in the absence of compound.

Carageenan induced hyperalgesia and Freund's complete adjuvant (CFA)induced hyperalgesia are models of inflammatory pain (see, e.g., Walkeret al. J Phannacol Exp Ther (2003) 304:56-62; McGaraughty et al. Br JPharmacol (2003) 140:1381-1388; Honore et al. J Pharmacol Exp Ther(2005) 314:410-421). Compounds that antagonize TRPA1 can be administeredto carrageenan or CFA challenged animals to assess whether they diminishcold, mechanical or heat hypersensitivity in comparison to that observedin the absence of compound. In addition, the ability of compounds thatantagonize TRPA1 function to diminish cold and/or mechanicalhypersensitivity can also be assessed in these models. Typically, thecarrageenan induced hyperalgesia model is believed to mimic acuteinflammatory pain and the CFA model is believed to mimic chronic painand chronic inflammatory pain.

Exemplary models of inflammatory pain include the rat model ofintraplantar bradykinin injection. Briefly, the baseline thermalsensitivity of the animals is assessed on a Hargreave's apparatus. TRPA1blockers are then administered systemically. Bradykinin is subsequentlyinjected into the paw and a hyperalgesia is allowed to develop. Thermalescape latency is then measured at multiple time points over the nextfew hours (Chuang et al., 2001; Vale et al., 2004).

Inflammation is often an important contributing factor to pain. As such,it is useful to identify compounds that act as anti-inflammatories. Manycompounds that reduce neural activity also prevent neurogenicinflammation. To measure inflammation directly, the volume of a rat pawcan be assessed using a plethysmometer. After baseline measurement istaken, carrageenan can be injected into the paw and the volume can bemonitored over the course of hours in animals that have been treatedwith vehicle or drug. Drugs that reduce the paw swelling are consideredto be anti-inflammatory.

Migraines are associated with significant pain and inability to completenormal tasks. Several models of migraine exist including the ratneurogenic inflammation model (see e.g., Buzzi et al Br J Phannacol(1990) 99:202-206) and the Burstein Model (see, e.g., Strassman et al.,Nature (1996) 384: 560-564).

The Bennett model uses prolonged ischemia of the paw to mirror chronicpain (see, e.g., Xanthos et al. J Pain (2004) 5: S1). This provides ananimal model for chronic pain including post-operative pain, complexregional pain syndrome, and reflex sympathetic dystrophy. Prolongedischemia induces behavioral changes in the animals includinghyperalgesia to mechanical stimuli, sensitivity to cold, pain behaviors(e.g., paw shaking, licking, and/or favoring), and hyperpathia.Compounds that antagonize TRPA1 can be administered to challengedanimals to assess whether they diminish any or all of these behaviors incomparison to that observed in the absence of compound. Similarexperiments can be conducted in a thermal injury or UV-burn model whichcan be used to mimic post-operative pain.

Additional models of neuropathic pain include central pain models basedon spinal cord injury. Chronic pain is generated by inducing a spinalcord injury, for example, by dropping a weight on a surgically exposedarea of spinal cord (e.g., weight-drop model). Spinal cord injury canadditionally be induced by crushing or compressing the spinal cord, bydelivering neurotoxin, using photochemicals, or by hemisecting thespinal cord.

Additional models of neuropathic pain include peripheral nerve injurymodels. Exemplary models include, but are not limited to, the neuromamodel, the Bennett model, the Seltzer model, the Chung model (ligationat either L5 or L5/L6), the sciatic cryoneurolysis model, the inferiorcaudal trunk resection model, and the sciatic inflammatory neuritismodel. Id.

Exemplary models of neuropathic pain associated with particular diseasesare also available. Diabetes and shingles are two diseases oftenaccompanied by neuropathic pain. Even following an acute shinglesepisodes, some patients continue to suffer from postherpetic neuralgiaand experience persistent pain lasting years. Neuropathic pain caused byshingles and/or postherpetic neuralgia can be studied in thepostherpetic neuralgia model (PHN). Diabetic neuropathy can be studiedin diabetic mouse models, as well as chemically induced models ofdiabetic neuropathy.

As outlined above, cancer pain may have any of a number of causes, andnumerous animal models exist to examine cancer pain related to, forexample, chemotherapeutics or tumor infiltration. Exemplary models oftoxin-related cancer pain include the vincristine-induced peripheralneuropathy model, the taxol-induced peripheral neuropathy model, and thecisplatin-induced peripheral neuropathy model. An exemplary model ofcancer pain caused by tumor infiltration is the cancer invasion painmodel (CIP). Id.

Primary and metastatic bone cancers are associated with tremendous pain.Several models of bone cancer pain exist including the mouse femur bonecancer pain model (FBC), the mouse calcaneus bone cancer pain model(CBC), and the rat tibia bone cancer model (TBC). Id.

An additional model of pain is the formalin model Like the carrageenanand CFA models, the formalin model involves injection of an irritantintradermally or intraperitoneally into an animal. Injection offormalin, a 37 percent solution of formaldehyde, is the most commonlyused agent for intradermal paw injection (the formalin test). Injectionof a 0.5 to 15 percent solution of formalin (usually about 3.5%) intothe dorsal or plantar surface of the fore- or hindpaw produces abiphasic painful response of increasing and decreasing intensity forabout 60 minutes after the injection. Typical responses include the pawbeing lifted, licked, nibbled, or shaken. These responses are considerednociceptive. The initial phase of the response (also known as the EarlyPhase), which lasts 3 to 5 minutes, is probably due to direct chemicalstimulation of nociceptors. This is followed by 10 to 15 minutes duringwhich animals display little behavior suggestive of nociception. Thesecond phase of this response (also known as the Late Phase) startsabout 15 to 20 minutes after the formalin injection and lasts 20 to 40minutes, initially rising with both number and frequency of nociceptivebehaviors, reaching a peak, then falling off. The intensities of thesenociceptive behaviors are dependent on the concentration of formalinused. The second phase involves a period of sensitization during whichinflammatory phenomena occur. The two phases of responsiveness toformalin injection makes the formalin model an appropriate model forstudying nociceptive and acute inflammatory pain. It may also model, insome respects, neuropathic pain.

In addition to any of the foregoing models of chronic pain, compoundsthat antagonize TRPA1 function can be tested in one or more models ofacute pain (see, e.g., Valenzano et al. (2005) Neuropharmacology48:658-672). Regardless of whether compounds are tested in models ofchronic pain, acute pain, or both, these studies are typically (thoughnot exclusively) conducted, for example, in mice, rats, or guinea pigs.Additionally, compounds can be tested in various cell lines that providein vitro assays of pain.

Many individuals seeking treatment for pain suffer from visceral pain.Animal models of visceral pain include the rat model of inflammatoryuterine pain (see, e.g., Wesselmann et al., Pain (1997) 73:309-317),injection of mustard oil into the gastrointestinal tract to mimicirritable bowel syndrome (see, e.g., Kimball et al., (2005)Am J PhysiolGastrointest Liver Physiol, 288(6):G1266-73), injection of mustard oilinto the bladder to mimic overactive bladder or bladder cystitis (see,e.g., Riazimand (2004), BJU Int 94:158-163). The effectiveness of aTRPA1 compound can be assessed by a decrease in writhing,gastrointestinal inflammation or bladder excitability.

For testing the efficacy of TRPA1 antagonists for the treatment ofcough, experiments using the conscious guinea pig model of cough can bereadily conducted (see, e.g., Tanaka and Maruyama (2003) J Pharmacol Sci93:465-470; McLeod et al. (2001) Br J Pharmacol 132: 1175-1178).Briefly, guinea pigs serve as a useful animal model for cough because,unlike other rodents such as mice and rats, guinea pigs actually cough.Furthermore, guinea pig coughing appears to mimic human coughing interms of the posture, behavior, and appearance of the coughing animal.

To induce cough, conscious guinea pigs are exposed to an inducing agentsuch as citric acid or capsaicin. The response of the animal is measuredby counting the number of coughs. The effectiveness of a coughsuppressing agent, for example a compound that inhibits TRPA1, can bemeasured by administering the agent and assessing the ability of theagent to decrease the number of coughs elicited by exposure to citricacid, capsaicin, or other similar cough-inducing agent. In this way,TRPA1 inhibitors for use in the treatment of cough can be readilyevaluated and identified.

Additional models of cough may also include the unconscious guinea pigmodel (see, e.g., Rouget et al. (2004) Br J Pharmacol 141: 1077-1083).Either of the foregoing models can be adapted for use with other animalscapable of coughing. Exemplary additional animals capable of coughinginclude cats and dogs.

Compounds of the invention may be tested in multiple models of asthma.One example is the murine ovalbumin model of asthma (see, e.g., CaceresA I et al., Proc Natl Acad Sci USA. (2009) 106(22):9099-104). In thismodel, ovalbumin is injected into the intraperitoneal cavity severaltimes over 2 weeks. Sometime in the third week, animals are challengedwith intranasal ovalbumin an airway hyperresponsiveness, inflammationand inflammatory cytokine production may be measured. Compounds aredosed during the challenge phase of the model. Trpa1 knock-out mice maybe substituted into the above models as reported by Caceres et al.

An example of a large animal model of asthma the conscious allergicsheep model as described in Abraham, W. M. et al. may be used to assesseffects of compounds on the antigen-induced late stage response ofasthma (Abraham W M., Am J Respir Crit Care Med (2000) 162(2):603-11).Briefly, baseline airway responsiveness is measured by plethysmograph inconscious sheep prior to a nebulized administration of Ascaris suumextract to induce asthma. After baseline readings are captured, animalsare challenged with a nebulized dose of Ascaris suum. Antigensensitivity is determined by decrease in pulmonary flow resistance frombaseline. Once animals demonstrate antigen-sensitivity, test compoundsmay be administered and additional pulmonary flow resistance readingscaptured to assess changes airway responsiveness. Models in the horseand beagle dog are sometimes also used.

Additional models may include the Brown Norway rat model and theC57BL/6J mouse model of asthma as described in Raemdonck et al.(Raemdonck K et al., Thorax (2012) January; 67(1):19-25). Briefly BrownNorway rats and C57BL/6J mice may be sensitized and challenged withaerosol delivered ovalbumin. Once sensitivity is confirmed by a decreasein lung function as measured by whole body plethysmograph readings,compounds of the invention may be administered. Visual and audible signsof respiratory distress including wheezing may also be present.

Dermatitis

Multiple mouse models of dermatological disease currently exist. Forexample, Liu et al. describe multiple oxazolone and urushiol-inducedcontact dermatis models (see, e.g., Liu B et al., FASEB J. (2013)27(9):3549-63). Briefly, Trpa1 knock-out mice receive topicaladministrations of oxazolone or urushiol to induce dermatitis and itchresponses. Epidermis thickness may also be measured by taking earpunches and measurements of challenged areas compared with untreatedears. In vivo treatment compounds may be determined by administeringcompounds to the animals prior to or after ozazolone or urushioltreatments. Scratching behaviors are recorded by video cameraspositioned above observation chambers. Observers blind to treatmentgroups record the time animals spend scratching over the course ofthirty minutes.

An alternative mouse model of dry-skin evoking itch involvesadministration of acetone, ether, and water to the mouse as reported byWilson et al. (Wilson S R et al., J Neurosci (2013) 33(22):9283-94) Inthis model, the area to be treated is shaved and mice receive topicaladministration of acetone and ether twice daily on the area to beobserved, e.g. cheek or caudal back. In vivo efficacy of treatmentcompounds may be determined by administering compounds to the animalsprior to or after acetone and ether administration. Scratching behavioris recorded by camera for a period of 20 minutes and quantified byobservers blind to treatment groups.

In addition, pruritus may be induced by direct injection of an agentthat causes itch. Examples of these agents may be found in Akayimo andCarstens, 2013. Some examples are: chloroquine (Wilson et al., 2011),bile acids, TSLP (Wilson et al., 2013), and IL-31 (Cevikbas et al.,2014). Typically scratching bouts in a defined period are recorded by anobserved blinded to treatment group.

Numerous rodent models of incontinence exist. These include models ofincontinence induced by nerve damage, urethral impingement andinflammation. Models of urethral impingement include the rat bladderoutflow obstruction model (see, e.g., Pandita, R K, and Andersson K E. JUrol (1999) 162: 943-948). Inflammatory models include injection ofmustard oil into the bladder.

To test the effectiveness of a TRPA1 inhibitor compound in treatingincontinence, varying concentrations of compound (e.g., low, medium, andhigh concentration) can be administered to rats following surgicalpartial bladder outlet obstruction (BOO). Efficacy of the varying dosesof TRPA1 inhibitory compound can be compared to controls administeredexcipients alone (sham control). Efficacy can further be compared torats administered a positive control, such as atropine. Atropine isexpected to decrease bladder over-activity following partial bladderoutlet obstruction in the BOO model. Note that when testing compounds inthe BOO model, compounds can be administered directly to the bladder orurethra (e.g., by catheter) or compounds can be administeredsystemically (e.g., orally, intraveneously, intraperitoneally, etc).

Several rat models of pancreatitic pain have recently been described(Lu, 2003, Anesthesiology 98(3):734-740; Winston et al., (2003) Journalof Pain 4(6): 329-337). Lu et al. induced pancreatitis by systemicdelivery of dibutylin dichloride in rats. Rats showed an increase inwithdrawal events after von Frey filament stimulation of the abdomen anddecreased withdrawal latency after thermal stimulation during a periodof 7 days. The pain state induced in these animals was alsocharacterized by increased levels of substance P in spinal cords (Lu, etal., 2003). To test the efficacy of a TRPA1 inhibitor in this model, aTRPA1 inhibitor can be administered following or concurrently withdelivery of dibutylin dichloride. Control animals can be administered acarrier or a known pain reliever. Indicia of pain can be measured.Efficacy of a TRPA1 inhibitor can be evaluated by comparing the indiciaof pain observed in animals receiving a TRPA1 inhibitor to that ofanimals that did not receive a TRPA1 inhibitor. Additionally, efficacyof a TRPA1 inhibitor can be compared to that of known pain medicaments.

The efficacy of von Frey filament testing as a means to measurenociceptive behavior was also shown by inducing pancreatitis by systemicL-arginine administration (Winston et al, 2003). The efficacy of a TRPA1inhibitor can similarly be tested following pancreatitis induced bysystemic L-arginine administration.

Lu et al. also described direct behavioral assays for pancreatic painusing acute noxious stimulation of the pancreas via an indwelling ductalcannula in awake and freely moving rats. These assays included cagecrossing, rearing, and hind limb extension in response tointrapancreatic bradykinin infusion. Intrathecal administration ofeither D-APV (NMDA receptor antagonist) or morphine alone partiallyreduced visceral pain behaviors in this model. Combinations of bothreduced pain behaviors to baseline. The efficacy of a TRPA1 inhibitorcan similarly be tested in this system.

Any of the foregoing animal models may be used to evaluate the efficacyof a TRPA1 inhibitor in treating pain associated with pancreatitis. Theefficacy can be compared to a no treatment or placebo control.Additionally or alternatively, efficacy can be evaluated in comparisonto one or more known pain relieving medicaments.

EXAMPLES General Procedures

All reactions were run under an inert atmosphere, generally nitrogen.All non-aqueous reactions were run using anhydrous solvents. Allreactions were stirred either with a magnetic stir bar or with overheadmechanical stirring. All saturated extraction solutions are assumed tobe aqueous (saturated NH₄Cl for example). All drying agents areanhydrous. Drying organic solutions with a drying agent implies that thedrying agent was removed from the organic solution by filtration.Chromatography refers to column chromatography on silica gel.Preparative thin layer chromatography (TLC) was run on silica gelplates. Concentration of reaction mixtures implies concentration underreduced pressure and the use of a rotary evaporation instrument. Dryingof final products implies drying under high vacuum conditions.Sonication implies the use of an ultrasonic bath. All ¹H-NMR data wereobtained at 400 MHz. Mass spectra were obtained in positive ion mode andare reported as the protonated species MH⁺ unless otherwise indicated.

ABBREVIATIONS

DCM dichloromethaneDIC N,N′-diisopropylcarbodiimideDIPEA N,N′-diisopropylethylamineDMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideEA ethyl acetateEther diethyl etherh hoursHOAc acetic acidHOAT 1-hydroxy-7-azabenzotriazoleLAH lithium aluminum hydrideMeOH methanolmin minutesn-BuLi n-butyllithium

NMP N-methylpyrrolidinone

Pd/C palladium on activated carbon, generally 10% palladium loadPE petroleum etherRT room temperatureTBAI tetrabutylammonium iodideTEA triethylamineTFA trifluoroacetic acidTLC thin layer chromatographyTHF tetrahydrofuran

Preparation of Synthetic Intermediates Preparation 1(2S)-2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid

Step 1 (R)-methyl 2-(methylsulfonyloxy)propanoate

A solution of (R)-methyl 2-hydroxypropanoate (30 g, 0.28 mol) and TEA(80 mL, 0.56 mol) in DCM (300 mL) was chilled to 0° C. andmethanesulfonyl chloride (33.6 mL, 0.42 mol) was added dropwise at 0° C.over 1 h. The mixture was stirred at 10-20° C. for 1.5 h. The resultingmixture was quenched with ice-water (100 mL). The organic layer wasseparated, washed with water (2×50 mL) and brine, dried over Na₂SO₄ andconcentrated to afford the crude product (R)-methyl2-(methylsulfonyloxy)propanoate (50 g, 95.2%) as brick red oil which wasused without purification.

Step 2 (2S)-methyl2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetra-hydro-1H-purin-7(2H)-yl)propanoate

To a suspension of 1,3-dimethyl-3,4,5,7-tetrahydro-1H-purine-2,6-dione(112 g, 0.62 mol) and K₂CO₃ (171 g, 1.24 mol, 2 eq) in DMF (2.2 L) at18° C. was added (R)-methyl 2-(methylsulfonyloxy)propanoate (226 g, 1.24mol). The mixture was stirred at 18° C. overnight; then it was quenchedwith saturated NH₄Cl (2 L). The resulting mixture was extracted with DCM(3×1 L). The combined organic phase was washed with water (5×500 mL) andbrine, dried over Na₂SO₄ and concentrated. The residue was taken up inDCM and extracted with 6N HCl (2×200 mL). The combined aqueous phase wasback-extracted with DCM (2×50 mL). The combined organic phase was driedover Na₂SO₄ and concentrated to give the desired product as a pale brownoil (65 g, 39.3%) which was used without further purification. MH⁺ 267.

Step 3(2S)-2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid

To a solution of (2S)-methyl2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetra-hydro-1H-purin-7(2H)-yl)propanoate(39 g, 145 mmol) in dioxane (400 mL) was added 6N HCl (200 mL). Themixture was refluxed for 3 h, cooled to room temperature and thenconcentrated to remove the dioxane and most of the aqueous phase. Theresidue was triturated in water (70 mL) and filtered. The solid wascollected by filtration to give the title product (17.3 g, ee: 99%*).The filtrate was concentrated to dryness and the residue was purified bychromatography eluting with DCM/MeOH (40/1 to 15/1) to give anadditional product (3.2 g, ee: 95%*). Total yield was 55.1%. ¹H NMR(DMSO-d₆) δ 13.28 (s, 1H), 8.21 (s, 1H), 5.47 (q, J=7.4 Hz, 1H), 3.44(s, 3H), 3.21 (s, 3H), 1.76 (d, J=7.4 Hz, 3H). MH⁺ 253. * Chiral HPLCdetails: Chiralcel AD Column, 250*4.6 mm, 10 um. Mobile phase: hexane(0.1% TFA)/IPA (0.1% TFA) 70/30.

Preparation 2 5,5-dimethylpyrrolidin-2-one hydrochloride

Step 1 methyl 4-methyl-4-nitropentanoate

To a solution of 2-nitropropane (5.06 g, 56.84 mmol) in dioxane (3 mL)was added Triton B (0.55 mL, 40% aqueous). The reaction was warmed to70° C. and methyl acrylate (4.78 g, 55.58 mmol) was added dropwise.After addition, the reaction was heated at 100° C. for 4 hrs. Thereaction was cooled to RT, 1N HCl (2 mL) was added, the resultingmixture was partitioned between EA and water. The combined organic layerwas washed with brine, dried over Na₂SO₄, and concentrated to afford thecrude product (10 g, 100%) as an oil. ¹H NMR (CDCl₃) δ 3.68 (s, 3H),2.35-2.31 (m, 2H), 2.27-2.23 (m, 2H), 1.60 (s, 6H).

Step 2 5,5-dimethylpyrrolidin-2-one

To a solution of NiCl₂ hexahydrate (0.67 g, 2.86 mmol) in MeOH (30 mL)was added NaBH₄ (0.33 g, 8.57 mmol) portionwise. The reaction wassonicated for 0.5 hr; then methyl 4-methyl-4-nitropentanoate (1.0 g,5.77 mmol) was added dropwise. Additional NaBH₄ (0.66 g, 17.14 mmol) wasadded portionwise. The resulting mixture was stirred at room temperatureovernight. The mixture was filtered through Celite and the filtrate wasconcentrated to one forth volume. The residue was partitioned betweenDCM and saturated NaHCO₃. The organic layer washed with brine, driedover Na₂SO₄, and concentrated to afford the crude product (0.35 g,53.7%) as an oil. MH⁺ 114.

Step 3 5,5-dimethylpyrrolidin-2-one hydrochloride

To a suspension of LAH (121 mg, 3.18 mmol) in THF (8 mL) was added5,5-dimethylpyrrolidin-2-one (0.3 g, 2.65 mmol) and the reaction washeated at 60° C. overnight. The reaction was cooled to 0° C. andcarefully quenched with water (0.2 mL) followed by 15% NaOH (0.2 mL).The mixture was filtered through celite. Concentrated hydrochloride acidwas added to the filtrate. This mixture was concentrated to afford thecrude product (0.2 g, 75.5%) as a white solid, which was used withoutpurification. MH⁺ 100.

Preparation 32′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine

Step 1 5-bromo-2-(2,2-dimethylpyrrolidin-1-yl)pyrimidine

To a solution of 5-bromo-2-chloropyrimidine (2.3 g, 11.9 mmol) and K₂CO₃(6.6 g, 47.6 mmol) in DMF (20 mL) was added a solution of2,2-dimethylpyrrolidine (2.26 g, 16.7 mmol) in DMF (4 mL) at RT. Theresulting reaction mixture was stirred at 50° C. for two days. Thereaction was poured into ice water with stirring. The precipitate wascollected to give crude5-bromo-2-(2,2-dimethylpyrrolidin-1-yl)pyrimidine (2.3 g, 76.6%) as alight yellow solid, which was used for next step without any furtherpurification. MH⁺ 256.

Step 22-(2,2-dimethylpyrrolidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine

A mixture of 5-bromo-2-(2,2-dimethylpyrrolidin-1-yl)pyrimidine (17.6 g,68.9 mmol), bis(pinacolato)diboron (24.5 g, 96.5 mmol), and KOAc (13.5g, 0.14 mol) in 1,4-dioxane (320 mL) was added Pd(PPh₃)₂Cl₂ (2.4 g, 3.45mmol). The mixture was stirred at 80° C. for 20 h. The reaction wascooled to RT, poured into ice-water, and extracted with EA (4×200 mL).The combined organic layer was washed with brine, dried over Na₂SO₄,concentrated to give a dark residue. The residue was purified bychromatography eluting with PE/EA (40:1) to give2-(2,2-dimethylpyrrolidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine(11.5 g, 49% over two steps) as a yellow solid. ¹H NMR (DMSO-d₆) δ 8.58(s, 2H), 3.69 (m, 2H), 1.92 (m, 4H), 1.55 (s, 6H), 1.33 (s, 12H).

Step 3 2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine

To a mixture of2-(2,2-dimethylpyrrolidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine(9.5 g, 31 mmol) in 1,4-dioxane (140 mL) was added4-amino-2-chloropyrimidine (4.5 g, 34.5 mmol) and 2M K₂CO₃ (20.4 mL,40.7 mmol). The orange mixture was degassed with N₂; then Pd(PPh₃)₄(3.65 g, 3.1 mmol) was added. The reaction was stirred at 80° C.overnight. The reaction was cooled to RT, poured into water andextracted with EtOAc (3×150 mL). The combined organic layer was washedwith brine, dried over Na₂SO₄, and concentrated. The residue waspurified by chromatography eluting with PE:EA (1:1) to afford compound2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (8.96g, >100%) as a yellow solid. MH+ 271.

Preparation 4(S)-2-(2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine

Step 1 (S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine

A mixture of (S)-2-(trifluoromethyl)pyrrolidine hydrochloride (40 g,0.23 mol), K₂CO₃ (94.6 g, 0.68 mol) and 5-bromo-2-chloropyrimidine (48g, 0.25 mol) in DMF (200 mL) was stirred at 100° C. for 24 hr, thenN₁,N₂-dimethylethane-1,2-diamine (4 mL) was added and the reaction wasstirred for another 2 h to consume excess 5-bromo-2-chloropyrimidine.The reaction was quenched with water (400 mL), and extracted with EA(3×500 mL). The combined organic phase was washed with 10% aqueous LiCl,dried over Na₂SO₄ and concentrated. The residue was purified bychromatography eluting with PE:EA (50:1) to afford(S)-5-bromo-2-(2-(trifluoromethyl) pyrrolidin-1-yl)pyrimidine (50 g,74%) as a white solid. ¹H NMR (DMSO-d₆) δ 8.54 (s, 2H), 4.90-4.94 (m,2H), 3.56-3.58 (m, 2H), 2.02-2.16 (m, 4H). MH⁺ 296.

Step 2 (S)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-ylboronicacid

A solution of(S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine (50 g, 0.17mol) and triisopropyl borate (44.4 g, 0.23 mol) in THF (400 mL) wascooled to −78° C. and n-BuLi (105 mL, 2.4 M in hexane) was addeddropwise. The reaction was stirred 2 h at −78° C. The reaction wasquenched with water (150 mL) and allowed to warm to RT. The reaction wasconcentrated to leave the aqueous phase. The aqueous phase was extractedwith ether (2×50 mL) to remove impurities (product in aqueous layer).The pH was adjusted to 5 with 6 N HCl and then it was extracted with EA(3×100 mL). The combined organic phase was dried over Na₂SO₄ andconcentrated to afford (S)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-ylboronic acid (45 g, quantitative yield) as an off-whitesolid. MH⁺ 262.

Step 3(S)-2-(2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine

To a mixture of(S)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-ylboronic acid(9.5 g, 36.4 mmol), 2-chloropyrimidin-4-amine (4.3 g, 33.1 mmol) andNa₂CO₃ (7.0 g, 66.2 mmol) in dioxane (105 mL) and water (35 mL) wasadded Pd(PPh₃)₄ (3.8 mg, 3.31 mmol). The mixture was degassed withnitrogen and then stirred at 110° C. for 3 h. The reaction was cooledand filtered through Celite. The filtrate was partitioned with EA (300mL) and water (150 mL). The organic phase was washed with brine (100mL), dried over Na₂SO₄ and concentrated. The residue was purified bychromatography eluting with DCM/MeOH (100:1 to 80:1 to 70:1) to give(S)-2-(2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine(8 g, 78%) as a white solid. ¹H-NMR (CDCl₃) δ 9.16 (s, 2H), 8.13-8.14(d, J=10 Hz, 1H), 6.97 (s, 2H), 6.34-6.35 (d, J=6 Hz, 1H), 5.09-5.13 (m,1H), 3.67-3.72 (m, 2H), 2.06-2.21 (m, 4H). MH⁺ 311.

Preparation 5(R)-2-(2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine

The title compound was prepared using the method of preparation 4. MH⁺311

Preparation 6(2S)-2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid

Step 1 (2S)-methyl2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoate

To a suspension of 1-methyl-3,4,5,7-tetrahydro-1H-purine-2,6-dione(6.904 g, 41.5 mmol) and K₂CO₃ (5.734 g, 41.5 mmol) in DMF (150 mL) at50° C. was added (R)-methyl 2-(methylsulfonyloxy)propanoate (5.818 g,32.0 mmol). The reaction was stirred at 50° C. overnight, then quenchedwith saturated NH₄Cl (2 L). The resulting mixture was extracted with EA(3×200 mL). The combined organic phase was washed with water (5×500 mL)and brine. The organic phase was dried over Na₂SO₄ and concentrated. Theresidue was purified by chromatography (0-3% MeOH:DCM) to give the titleproduct as a white solid (1.649 g, 20%). MH⁺ 253.

Step 2(2S)-2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid

To a mixture of (2S)-methyl2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoate(96.9 mg, 0.38 mmol) in dioxane (3 mL) was added 6N HCl (2 mL). Thereaction was refluxed for 3 h, cooled to room temperature andconcentrated to give the white solid product (92 mg, 100%); MH⁺ 239.

Preparation 7(S)-2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid

Step 1 (S)-methyl2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoate

To a solution of (2S)-methyl2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoate(600 mg, 2.38 mmol) in DMF (2 ml) at RT was added sodium2-chloro-2,2-difluoroacetate (508 mg, 3.33 mmol) followed by Cs₂CO₃ (229mg 3.81 mmol). The reaction was heated at 60° C. for 12 h. The reactionwas cooled to RT, diluted with cold water and extracted with EA twice.The combined organic layer was washed with brine, dried over MgSO₄ andconcentrated. The residue was purified by chromatography eluting withMeOH:DCM (0-3%) to give (S)-methyl2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoate(164 mg, 23%) as colorless oil. MH⁺ 303.

Step 2(S)-2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid

To a mixture of (S)-methyl2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoate(64 mg, 0.21 mmol) in dioxane (1 mL) was added 6N HCl (1 mL). Thereaction was refluxed for 3 h, cooled to RT and then concentrated. Theprecipitate was collected to give the white solid product (61 mg, 100%).MH⁺ 289.

Preparation 8 2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-amine

Step 1 5-bromo-2-(3,3-difluoroazetidin-1-yl)pyrimidine

A sealed tube was charged with 5-bromo-2-chloropyrimidine (450 mg, 2.3mmol), 3,3-difluoroazetidine hydrochloride (275.1 mg, 2.1 mmol), K₂CO₃(589.3 mg, 4.3 mmol), and DMF (3 mL). The tube was sealed and stirred at130° C. for 2 h. The reaction was cooled to RT and poured into water (4mL). The solid was collected by filtration and dried to give5-bromo-2-(3,3-difluoroazetidin-1-yl)pyrimidine (300 mg, 51.4%) as awhite solid. MH⁺ 250.

Step 2 (2-(3,3-difluoroazetidin-1-yl)pyrimidin-5-yl)boronic acid

To a solution of 5-bromo-2-(3,3-difluoroazetidin-1-yl)pyrimidine (300mg, 1.2 mmol) and triisopropyl borate (0.4 mL, 1.8 mmol) in THF (6 mL)was added n-BuLi (0.6 mL, 2.4 M in hexane, 1.5 mmol) dropwise at −78° C.The mixture was stirred at −78° C. for 2 h. The reaction was quenchedwith water and warmed to RT. The solvent was concentrated and theresidual aqueous layer was extracted with ether (2×10 mL). The aqueouslayer was adjusted to pH 6 with 1N HCl and extracted with EA (3×10 mL).The combined organic layer was washed with brine, dried over Na₂SO₄, andconcentrated to give the product (170 mg, 65.6%) as a white solid. MH⁺216.

Step 3 2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-amine

A mixture of (2-(3,3-difluoroazetidin-1-yl)pyrimidin-5-yl)boronic acid(170.0 mg, 0.8 mmol), 4-amino-2-chloropyrimidine (102.4 mg, 0.8 mmol),Pd(PPh₃)₂Cl₂ (56.2 mg, 0.08 mmol) and Na₂CO₃ (167.5 mg, 1.6 mmol) in1,4-dioxane (5 mL) and water (1 mL) was degassed with nitrogen andstirred at 90° C. overnight. The resulting mixture was cooled to RT andpoured into EA. The organic phase was separated, washed with water andbrine, dried over Na₂SO₄ and concentrated. The residue was dissolved inether. An insoluble residue was removed by filtration and the filtratewas concentrated to give2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-amine (130 mg,62.3%) as a white solid. MH⁺ 265.

Preparation 92-chloro-N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide

To a solution of2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-amine (60 mg, 0.2mmol) in DMF (2 mL) was added dropwise 2-chloroacetyl chloride (0.03 mL,0.34 mmol) at 0° C. The reaction was stirred at RT for 2 h and then itwas poured into EA. This organic phase was washed with water and brine,dried over Na₂SO₄, and concentrated to give2-chloro-N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(50 mg, 64.7%) as a yellow solid. MH⁺ 341.

Preparation 10 (2-(4,4-difluoropiperidin-1-yl)pyrimidin-5-yl)boronicacid

Step 1 5-bromo-2-(4,4-difluoropiperidin-1-yl)pyrimidine

A sealed tube were charged with 5-bromo-2-chloropyrimidine (633.3 mg,3.3 mmol), 4,4-difluoropiperidine hydrochloride (472.8 mg, 3.0 mmol),K₂CO₃ (829.3 mg, 6.0 mmol) and DMF (4 mL). The tube was sealed andstirred at 130° C. for 2 h; then it was cooled to RT and poured intowater (5 mL). The solid precipitate was collected and dried to give5-bromo-2-(4,4-difluoropiperidin-1-yl)pyrimidine (640 mg, 77%) as awhite solid. MH⁺ 278.

Step 2 (2-(4,4-difluoropiperidin-1-yl)pyrimidin-5-yl)boronic acid

To a solution of 5-bromo-2-(4,4-difluoropiperidin-1-yl)pyrimidine (640mg, 2.3 mmol) and triisopropyl borate (0.8 mL, 3.5 mmol) in THF (8 mL)was added n-BuLi (2 mL, 2.4 M in hexane, 1.5 mmol) dropwise at −78° C.The mixture was stirred at −78° C. for 2 h. This reaction was quenchedwith water and allowed to warm to RT. The reaction was concentrated andthe residual aqueous mixture was extracted with ether (2×10 mL). Theaqueous phase was adjusted to pH 6 with 1N HCl and extracted with EA(3×10 mL). The combined organic phase was washed with brine, dried overNa₂SO₄, and concentrated to give(2-(4,4-difluoropiperidin-1-yl)pyrimidin-5-yl)boronic acid (420 mg,74.8%) as a white solid. MH⁺ 244.

Preparation 11 2-chloro-N-(2-chloropyrimidin-4-yl)acetamide

To a mixture of 4-amino-2-chloropyrimidine (2.0 g, 15.4 mmol) and DMF(25 mL) was added dropwise 2-chloroacetyl chloride (0.03 mL, 0.34 mmol)at 0° C. The reaction was stirred at RT overnight and then it was pouredinto EA. The organic phase was washed with water and brine, dried overNa₂SO₄, and concentrated. The residue was triturated with DCM and thesolids were collected to give2-chloro-N-(2-chloropyrimidin-4-yl)acetamide (1.3 g, 20.5%) as a yellowsolid. MH⁺ 206.

Preparation 12N-(2-chloropyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide

A mixture of 2-chloro-N-(2-chloropyrimidin-4-yl)acetamide (1.1 g, 5.4mmol), 1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (966.3 mg, 5.4 mmol),K₂CO₃ (1.1 g, 8.1 mmol), and TBAI (198.2 mg, 0.5 mmol) in DMF (20 mL)was stirred at 90° C. for 10 min. The reaction was cooled to RT and thendiluted with EA. The resulting mixture was washed with water, saturatedNH₄Cl and brine, dried over Na₂SO₄ and concentrated. The residue wasrecrystallized from DCM to giveN-(2-chloropyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-acetamide(1.3 g, 69.4%) as a white solid. MH⁺ 350.

Preparation 13 3-azabicyclo[3.1.0]hexane hydrochloride

Step 1 3-benzyl-3-azabicyclo[3.1.0]hexane-2,4-dione

To a mixture of 3-oxabicyclo[3.1.0]hexane-2,4-dione (2.3 g, 20.5 mmol)in AcOH (30 mL) was added DMAP (150 mg) and benzylamine (2.2 mL, 20.5mmol). The mixture was stirred at 100° C. for 40 hr; then cooled to RT.The reaction was concentrated and the residue was dissolved in EA. Theorganic phase was washed with water and brine, dried over Na₂SO₄ andconcentrated. The residue was purified via chromatography eluting withPE:EA (8:1 to 5:1) to afford3-benzyl-3-azabicyclo[3.1.0]hexane-2,4-dione (3.7 g, 89.6%) as a whitesolid. MH⁺ 202.

Step 2 3-benzyl-3-azabicyclo[3.1.0]hexane

To a solution of 3-benzyl-3-azabicyclo[3.1.0]hexane-2,4-dione (2.0 g,10.0 mmol) in THF (30 mL) was added LAH (1.5 g, 40.0 mmol). Theresulting mixture was heated at reflux 4 h and then it was cooled to 0°C. The cold reaction mixture was carefully quenched with saturated NH₄Cland then it was filtered. The filtrate was concentrated to afford thetitle compound (1.5 g, 86.7%) as a clear oil. MH⁺ 174.

Step 3 3-azabicyclo[3.1.0]hexane hydrochloride

A mixture of 3-benzyl-3-azabicyclo[3.1.0]hexane (1.3 g, 7.5 mmol), 10%Pd/C (130 mg) and conc. HCl (0.63 mL, 7.5 mmol) in MeOH (20 mL) wasstirred at RT under an atmosphere of hydrogen (balloon) for 18 h. Thereaction was filtered through Celite and the filtrate was concentratedto give the title compound (850 mg, 95%) as a white solid. MH⁺ 84

Preparation 142′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine

Step 1 3-(5-bromopyrimidin-2-yl)-3-azabicyclo[3.1.0]hexane

A sealed tube was charged with 5-bromo-2-chloropyrimidine (671.7 mg, 3.5mmol), 3-azabicyclo[3.1.0]hexane hydrochloride (416.7 mg, 3.5 mmol),K₂CO₃ (967.5 mg, 7.0 mmol) and DMF (4 mL). The tube was sealed andstirred at 130° C. for 2 h. The reaction was cooled to RT and pouredinto cold water (4 mL). The solid that formed was collected and dried togive 3-(5-bromopyrimidin-2-yl)-3-azabicyclo[3.1.0]hexane (480 mg, 57.4%)as a white solid. MH⁺ 240.

Step 2 (2-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-5-yl)boronic acid

To a solution of 3-(5-bromopyrimidin-2-yl)-3-azabicyclo[3.1.0]hexane(480 mg, 2.0 mmol) and triisopropyl borate (0.7 mL, 3.0 mmol) in THF (6mL) was added n-BuLi (1.1 mL, 2.4 M in hexane, 2.6 mmol) dropwise at−78° C. The reaction was stirred at −78° C. for 2 hr and then it wasquenched with water and warmed to RT. The reaction was concentrated andthe aqueous residue was extracted with ether (2×20 mL). The aqueouslayer was separated, adjusted to pH 6 with 1N HCl and extracted with EA(3×20 mL). The combined organic phase was washed with brine, dried overNa₂SO₄, and concentrated to give the title product (200 mg, 48.5%) as awhite solid. MH⁺ 206.

Step 3 2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine

A mixture of (2-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-5-yl)boronicacid (150.0 mg, 1.2 mmol), 2-chloropyrimidin-4-amine (237.9 mg, 1.2mmol), Pd(PPh₃)₂Cl₂ (86.0 mg, 0.1 mmol) and Na₂CO₃ (245.9 mg, 2.3 mmol)in 1,4-dioxane (5 mL) and water (1 mL) was degassed with nitrogen andstirred at 80° C. overnight. The reaction was cooled to RT and pouredinto EA. The organic phase was separated, washed with water and brine,dried over Na₂SO₄ and concentrated. The residue was dissolved in ether.An insoluble residue was removed by filtration and the filtrate wasconcentrated to give2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine (100 mg,33.8%) as a white solid. MH⁺ 255.

Preparation 15N-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-yl)-2-chloroacetamide

To a solution of2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine (40 mg,0.2 mmol) in DMF (2 mL) was added dropwise 2-chloroacetyl chloride (0.02mL, 0.3 mmol) at 0° C. The reaction was stirred at RT for 2 h, thenpoured into EA. The organic layer was extracted with water and brine,dried over Na₂SO₄, and concentrated to giveN-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-yl)-2-chloroacetamide (50 mg, 96.2%) as a yellow solid. MH⁺ 329.

Preparation 162′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine

Step 1 5-bromo-2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyramidine

A sealed tube was charged with 5-bromo-2-chloropyrimidine (441.4 mg, 2.3mmol), 3-(trifluoromethyl)pyrrolidine hydrochloride (402.6 mg, 2.1mmol), K₂CO₃ (635.8 mg, 4.6 mmol) and DMF (3 mL). The tube was sealedand stirred at 130° C. for 2 h; then it was poured into water (4 mL).The solid was collected and dried to give5-bromo-2-(3,3-difluoroazetidin-1-yl)pyrimidine (500 mg, 73.7%) as awhite solid. MH⁺ 296.

Step 2 (2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)boronicacid

To a solution of5-bromo-2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyramidine (500 mg, 1.7mmol) and triisopropyl borate (0.6 mL, 2.5 mmol) in THF (6 mL) was addeddropwise n-BuLi (0.9 mL, 2.4 M in hexane, 2.2 mmol) at −78° C. Themixture was stirred at −78° C. for 2 h, then quenched with water andallowed to warm to RT. The reaction was concentrated and the aqueousresidue was extracted with ether (2×20 mL). The aqueous layer wasadjusted to pH 6 with 1N HCl and extracted with EA (3×20 mL). Thecombined organic phase was washed with brine, dried over Na₂SO₄, andconcentrated to give the title product (320 mg, 72.3%) as a white solid.MH⁺ 260.

Step 32′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine

A mixture of(2-(3-(trifluoromethyl)pyrrolidin-1-yl)pyrimidin-5-yl)boronic acid(320.0 mg, 1.2 mmol), 2-chloropyrimidin-4-amine (158.1 mg, 1.2 mmol),Pd(PPh₃)₂Cl₂ (86.0 mg, 0.1 mmol) and Na₂CO₃ (260.0 mg, 2.5 mmol) in1,4-dioxane (5 mL) and water (1 mL) was degassed with nitrogen andstirred at 90° C. overnight. The reaction was cooled to RT and pouredinto EA. The organic phase was washed with water and brine, dried overNa₂SO₄ and concentrated. The residue was dissolved in ether. Aninsoluble residue was removed by filtration and the filtrate wasconcentrated to give2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (200mg, 52.6%) as a white solid. MH⁺ 311.

Preparation 172-chloro-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide

To a solution of2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (93mg, 0.3 mmol) in DMF (2 mL) was added dropwise 2-chloroacetyl chloride(0.04 mL, 0.45 mmol) at 0° C. The reaction was stirred at RT for 2 h,then poured into EA. The organic phase was extracted with water andbrine, dried over Na₂SO₄, and concentrated to give2-chloro-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(100 mg, 86.4%) as a yellow solid. MH⁺ 387.

Preparation 182-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine

Step 1 2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-ylboronic acid

To a solution of 5-bromo-2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidine(550 mg, 1.95 mmol) and triisopropyl borate (383 mg, 2.74 mmol) in THF(5 mL) was added dropwise n-BuLi (1.1 mL, 2.4 M in hexane) at −78° C.The reaction was stirred at −78° C. for 2 h. The reaction was quenchedwith water (5 mL) and allowed to warm to RT. The reaction wasconcentrated and the aqueous residue was extracted with ether (2×2 mL).The aqueous layer was adjusted pH to 5 with 1N HCl and extracted with EA(3×5 mL). The combined organic phase was dried over Na₂SO₄ andconcentrated to afford2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-ylboronic acid (450 mg,93.7%) as an off-white solid. MH⁺ 248.

Step 22-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine

To a mixture of2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-ylboronic acid (450 mg,1.82 mmol), 2-chloropyrimidin-4-amine (213 mg, 1.65 mmol) and saturatedNa₂CO₃ (2.5 mL) in dioxane (10 mL) was added Pd(PPh₃)₂Cl₂ (58 mg, 0.08mmol) and degassed three times with nitrogen. The reaction was stirredat 90° C. overnight. The reaction was cooled to RT and filtered throughCelite. The filtrate was extracted with EA (2×4 mL). The combinedorganic phase was dried over Na₂SO₄ and concentrated. The residue waspurified via chromatography eluting with PE:acetone (3:1) to afford2-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine(350 mg, 71.4%) as a white solid. MH⁺ 297.

Preparation 192-chloro-N-(2-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-yl)acetamide

To a solution of2-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-amine(100 mg, 0.34 mmol) in DMF (2 mL) was added dropwise 2-chloroacetylchloride (0.045 mL, 0.51 mmol) at 0° C. The mixture was stirred at roomtemperature for 2 h, then poured into EA. The organic phase wasextracted with water and brine, dried over Na₂SO₄, and concentrated togive2-chloro-N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(70 mg, 56%) as a yellow oil. MH⁺ 373.1.

Preparation 20 2′-(4,4-difluoropiperidin-1-yl)-2,5′-bipyrimidin-4-amine

A mixture of (2-(4,4-difluoropiperidin-1-yl)pyrimidin-5-yl)boronic acid(142 mg, 0.58 mmol), 2-chloropyrimidin-4-amine (68.5 mg, 0.53 mmol),Pd(PPh3)₂Cl₂ (37.3 mg, 0.05 mmol), K₂CO₃ (146.8 mg, 1.06 mmol),1,4-dioxane (3 mL) and water (0.5 mL) was degassed with nitrogen andstirred at 90° C. for 2 h. The resulting mixture was cooled to RT andpoured into EA. The organic layer was separated, washed with water andbrine, dried over anhydrous Na₂SO₄ and concentrated. The residue waspurified with column chromatography eluting with DCM/MeOH (50:1) to give2′-(4,4-difluoropiperidin-1-yl)-[2,5′-bipyrimidin]-4-amine (15 mg, 10%)as a white solid. MH⁺ 293.

Preparation 21 (S)-2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-amine

Step 1 (S)-5-bromo-2-(2-methylpiperidin-1-yl)pyrimidine

To a solution of 5-bromo-2-chloropyrimidine (1.41 g, 7.35 mmol) in DMF(20 mL) was added (S)-2-methylpiperidine (800 mg, 8.08 mmol) and K₂CO₃(1.52 g, 11.03 mmol). The reaction was stirred at RT overnight. Thereaction was poured into ice water and extracted with EA (3×20 mL). Thecombined organic phase was washed with brine, dried over Na₂SO₄, andconcentrated. The residue was purified by chromatography eluting withPE:EA (80:1) to afford the title product (1.07 g, 56.9%) as a whitesolid. MH⁺ 240.

Step 2 (S)-2-(2-methylpiperidin-1-yl)pyrimidin-5-ylboronic acid

To a solution of (S)-5-bromo-2-(2-methylpiperidin-1-yl)pyrimidine (1.07g, 4.2 mmol) and triisopropyl borate (1.1 g, 5.87 mmol) in THF (20 mL)was added n-BuLi (5.25 mL, 1.6 M in hexane, 8.4 mmol) dropwise at −70°C. The reaction was stirred at −70° C. for 3 h, then was quenched withwater. The reaction was concentrated and the aqueous residue wasextracted with ether (2×20 mL). The aqueous phase was adjusted to pH 6with 1N HCl and extracted with EA (3×20 mL). The combined organic phasewas washed with brine, dried over Na₂SO₄, and concentrated to give thetitle product (900 mg, 96%) as a white solid, which was directly usedwithout purification. MH⁺ 222.

Step 3 (S)-2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-amine

A mixture of (S)-2-(2-methylpiperidin-1-yl)pyrimidin-5-ylboronic acid(752 mg, 3.4 mmol), 4-amino-2-chloropyrimidine (400 mg, 3.09 mmol),Pd(PPh₃)₂Cl₂ (216.0 mg, 0.3 mmol) and Na₂CO₃ (655 mg, 6.18 mmol) in1,4-dioxane (10 mL) and water (2.5 mL) was degassed with nitrogen andstirred at 80° C. for 2 h. The reaction was cooled to RT and partitionedbetween EA (20 mL) and water (15 mL). The organic phase was washed withwater and brine, dried over Na₂SO₄ and concentrated. The residue wasdissolved in ether. An insoluble residue was removed by filtration andthe filtrate was concentrated to give(S)-2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-amine (682 mg, 81.8%)as a white solid. MH⁺ 271.

Preparation 22(S)-2-chloro-N-(2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide

To a solution of(S)-2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-amine (400 mg, 1.48mmol) in DMF (8 mL) was added 2-chloroacetyl chloride (0.17 mL, 2.24mmol) dropwise at 0° C. The reaction was stirred at RT overnight, thenpoured into ice-water and extracted with EA (3×20 mL). The combinedorganic phase was washed with brine, dried over Na₂SO₄, and concentratedto afford the desired product (510 mg, 99%) as a yellow syrup. MH⁺ 347.

Preparation 232-chloro-N-(2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(LJ-262-64)

To a solution of2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (400 mg,1.48 mmol) in DMF (5 mL) was added dropwise 2-chloroacetyl chloride (251mg, 2.22 mmol) at 0° C. After stirring at RT overnight, the reactionmixture was partitioned with EA and water. The organic phase was washedwith water and brine, dried over Na₂SO₄, and concentrated to give2-chloro-N-(2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(500 mg, 97.4%) as a yellow solid. MH⁺ 347.

Preparation 24 (S)-2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine

Step 1 (S)-2-(2-methylpyrrolidin-1-yl)pyrimidin-5-ylboronic acid

To a solution of (S)-5-bromo-2-(2-methylpyrrolidin-1-yl)pyrimidine (7 g,30.8 mmol, prepared using the method described in WO2013/023102) andtriisopropyl borate (8.12 g, 43.2 mmol) in THF (70 mL) was added n-BuLi(28.9 mL, 1.6 M in hexane, 46.3 mmol) dropwise at −70° C. The reactionwas stirred at −70° C. for 3 h; then it was quenched with water. Thereaction was concentrated and the aqueous residue was extracted withether (2×20 mL). The aqueous layer was adjusted to pH 6 with 1N HCl andextracted with EA (3×20 mL). The combined organic phase was washed withbrine, dried over Na₂SO₄, and concentrated to give the title product(4.8 g, 75.3%) as a white solid. MH⁺ 208.

Step 2 (S)-2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine

A mixture of of (S)-2-(2-methylpyrrolidin-1-yl)pyrimidin-5-ylboronicacid (2.53 g, 12.23 mmol), 4-amino-2-chloropyrimidine (1.44 g, 11.12mmol), Pd(PPh₃)₂Cl₂ (432.0 mg, 0.6 mmol) and Na₂CO₃ (2.35 g, 22.24 mmol)in 1,4-dioxane (40 mL) and water (10 mL) was degassed with nitrogen andstirred at 80° C. for 2 h. The reaction was cooled to RT and poured intoEA. The organic phase was washed with water and brine, dried over Na₂SO₄and concentrated. The residue was taken up in ether. An insolubleresidue was removed by filtration and the filtrate was concentrated togive (S)-2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine (1.5 g,54%) as white solid. MH⁺ 257.

Preparation 25(S)-2-chloro-N-(2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide

To a solution of(S)-2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (256 mg, 1mmol) in DMF (5 mL) was added dropwise 2-chloroacetyl chloride (170 mg,1.5 mmol) at 0° C. After stirred at RT overnight, the reaction mixturewas partitioned with EA and water. The organic layer was washed withwater and brine, dried over Na₂SO₄, and concentrated to give(S)-2-chloro-N-(2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(280 mg, 84.1%) as a yellow solid. MH⁺ 333.

Preparation 262-chloro-N-(2′-((S)-2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

To a solution of(S)-2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine (800 mg, 3.12mmol) in DMF (15 mL) was added 2-chloropropanoyl chloride (0.33 mL, 3.43mmol) dropwise at 0° C. The reaction was stirred at RT overnight. Thereaction was poured into ice water and extracted with EA (3×20 mL). Thecombined organic phase was washed with brine, dried over Na₂SO₄, andconcentrated. The crude product was purified by chromatography elutingwith PE:EA (5:1) to give2-chloro-N-(2′-((S)-2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide(600 mg, 56%) as a viscous oil. MH⁺ 347.

Preparation 272-(3-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetic acid

A mixture of 3-methyl-1H-purine-2,6(3H,7H)-dione (15 g, 90 mmol), K₂CO₃(13.73 g, 99 mmol), DMF (451 mL), and ethyl 2-chloroacetate (9.62 mL, 90mmol) was heated at 90° C. for 0.5 h. The reaction was cooled to RT andwater (450 mL) was added. To the stirred solution was added LiOH (4.32g, 181 mmol) in water (100 mL). The reaction was stirred at RT for 1 h.The reaction was adjusted to pH 4 with 6N HCl. The precipitate thatformed was collected and dried to yield2-(3-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetic acid (18,670g, 92%) as a white powder. ¹H NMR (DMSO-d₆) δ 13.51 (s, 1H), 8.01 (s,1H), 5.03 (s, 2H), 3.36 (s, 3H).

Preparation 282-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)aceticacid

Step 1 tert-butyl2-(1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate

To a solution of 1-methylxanthine (9.049 g, 54.4 mmol) and potassiumcarbonate (8.258 g, 59.8 mmol) in DMF (200 mL), was added tert-butyl2-bromoacetate (8.03 mL, 54.4 mmol) dropwise. The reaction was stirredat 90° C. for 1 h and cooled to room temperature. The mixture was pouredinto water and acidified with HCl (6N, aq.) to pH 4. The mixture wasthen extracted with EA (200 mL×3). The combined organic layers werewashed with water (200 mL), dried over Na₂SO₄ and concentrated in vacuo.The crude product was purified by chromatography eluting with MaOH:DCM(2:100) to give tert-butyl2-(1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate (6.539 g,43%) as yellow solid. MH⁺ 281.

Step 2 tert-butyl2-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate

To a solution of tert-butyl2-(1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate (1.158 g,4.13 mmol) in DMF (20.66 mL) at RT was added K₂CO₃ (0.857 g, 6.20 mmol)and iodomethane-D3 (0.334 mL, 5.37 mmol). The reaction was heated at 65°C. for 2 h. Additional iodomethane-D3 (0.2 equiv) was added and heatingwas continued 1 h longer. The reaction was cooled to RT, diluted withwater and extracted three times with EA. The combined organic phase waswashed with brine, dried over MgSO₄ and concentrated. The residue waspurified by chromatography (0-100% EA:hexane) to afford tert-butyl2-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate(1.35 g, impure). MH⁺ 298. The impure product was used without furtherpurification.

Step 32-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)aceticacid

A solution of tert-butyl2-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetate(1.35 g, 4.54 mmol) in DCM (27.2 mL) was treated with TFA (18.16 mL).The reaction was stirred at RT for 2 h. The reaction was concentrated toan oil that was used without purification. MH⁺ 242.

Preparation 292′-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine

Step 1 3-(5-bromopyrimidin-2-yl)-6,6-difluoro-3-azabicyclo[3.1.0]hexane

A sealed tube was charged with 5-bromo-2-chloropyrimidine (748.5 mg, 3.9mmol), 6,6-difluoro-3-azabicyclo[3.1.0]hexane hydrochloride (604.6 mg,3.9 mmol), K₂CO₃ (1.1 g, 7.8 mmol) and NMP (3 mL). The mixture wasstirred at 130° C. for 3 h; then it was cooled to RT and poured intowater (4 mL). The solid was collected by filtration and dried undervacuum to give3-(5-bromopyrimidin-2-yl)-6,6-difluoro-3-azabicyclo[3.1.0]hexane (1.0 g,93.2% yield) as a white solid. MH⁺ 276.

Step 2(2-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-5-yl)boronicacid

To a solution of3-(5-bromopyrimidin-2-yl)-6,6-difluoro-3-azabicyclo[3.1.0]hexane (1.1 g,4.1 mmol) and (i-PrO)₃B (1.4 mL, 6.2 mmol) in THF (20 mL) was addedn-BuLi (3.9 mL, 1.6 M in hexane, 6.2 mmol) dropwise at −78° C. Thereaction was stirred at −78° C. for 2 h; then it was quenched with waterand warmed to RT. The solvent was removed under reduced pressure and theaqueous layer was washed with ether (2×50 mL). The aqueous layer wasthen adjusted to pH 6 with 1N HCl and extracted with EA (3×50 mL). Thecombined organic layers were washed with brine, dried over Na₂SO₄, andconcentrated to give2-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-5-yl)boronicacid (700 mg, 72.6% yield) as a white solid.

Step 32′-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine

A mixture of(2-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-5-yl)boronicacid (241.0 mg, 1.0 mmol), 2-chloropyrimidin-4-amine (129.0 mg, 1.0mmol), Pd(PPh₃)₄ (57.8 mg, 0.05 mmol) and K₂CO₃ (276.4 mg, 2.0 mmol) in1,4-dioxane (5 mL) and water (1 mL) was degassed and purged with N₂three times. The reaction was heated at 90° C. with stirring for 3 h.The resulting mixture was cooled to RT and poured into EA. The organicphase was separated, washed with water and brine, dried over Na₂SO₄ andconcentrated. The residue was purified with chromatography eluting withDCM:MeOH (50:1) to afford2′-(6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-amine(210 mg, 72.3% yield) as a white solid. MH⁺ 291.

Preparation 30(2R)-2-(1,3-dimethyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid

Step 1 (S)-methyl 2-(methylsulfonyloxy)propanoate

A solution of (S)-methyl 2-hydroxypropanoate (12.379 g, 119 mol) and TEA(17.4 mL, 125 mol) in DCM (100 mL) was chilled to 0° C. andmethanesulfonyl chloride (12.4 mL, 125 mol) was added dropwise at 0° C.over 1 h. The mixture was stirred at 20° C. for 1.5 h. The resultingmixture was quenched with ice-water (100 mL). The organic layer wasseparated, washed with water (2×50 mL) and brine, dried over Na₂SO₄ andconcentrated to afford the crude product (S)-methyl2-(methylsulfonyloxy)propanoate (20.940 g, 97%) as brown oil which wasused without purification.

Step 2(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid

To a suspension of 1,3-dimethyl-3,4,5,7-tetrahydro-1H-purine-2,6-dione(4.588 g, 25.5 mol) and K₂CO₃ (7.038 g, 51 mol, 2 eq) in DMF (500 mL) atRT was added (s)-methyl 2-(methylsulfonyloxy)propanoate (6.953 g, 38.2mol). The mixture was stirred at RT overnight, then quenched withsaturated NH₄Cl. The resulting mixture was extracted with DCM (3×300mL). The combined organic phase was washed with water and brine, driedover Na₂SO₄ and concentrated. The brown oil residue (8.633 g) wasdissolved in dioxane (10 mL). To the solution was added 6N HCl (aq. 10mL). The mixture was refluxed for 2 h, cooled to RT and thenconcentrated to remove the dioxane and most of the aqueous phase. Theresidue was purified with chromatography eluting with MeOH/DCM (0-10%)to afford(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (6.015 g, 93.6% yield) as white solid.

Synthesis of Compounds of Formula (I) Example 1(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

To a mixture of2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (4.2 g, 15.5mmol) and(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (3.56 g, 14.1 mmol) in DCM (72 mL) was added HOAT (1.92 g, 14.1mmol) at RT. The reaction was cooled to 0° C. and pyridine (2.23 g, 28.2mmol) and DIC (2.67 g, 21.2 mmol) were added. The reaction was warmed to25-28° C. and stirred overnight. The reaction mixture was quenched with0.5 N HCl. The mixture was added dropwise to n-hexane and theprecipitate that formed was collected and washed with MeOH to give(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2,2-dimethylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide(3 g, 19%). ¹H NMR (CDCl₃) δ 9.78 (s, 1H), 9.14 (s, 2H), 8.54 (d, J=5.6Hz, 1H), 7.91 (s, 1H), 7.76 (d, J=5.6 Hz, 1H), 5.83 (q, J=7.2 Hz 1H),3.77 (m, 2H), 3.63 (s, 3H), 3.50 (s, 3H), 1.96 (m, 7H), 1.60 (s, 6H).).

Example 2(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide(Compound 2)

To a mixture of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (2.44 g, 9.67 mmol) and(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine(3.3 g, 10.6 mmol) in DCM (48 mL) was added HOAT (1.3 g, 9.67 mmol) atRT. The mixture was cooled to 0° C. Pyridine (1.5 g, 19.3 mmol) wasadded dropwise over 30 min followed by dropwise addition of DIC (1.8 g,14.5 mmol). The reaction was stirred at 35° C. for 16 h; then it wasdiluted with DCM (100 mL). The mixture was extracted with saturatedNH₄Cl (50 mL, precooled to 0° C.) and brine, dried over Na₂SO₄, andconcentrated. The residue was purified by chromatography eluting firstwith EA:PE (3:2) and then DCM:MeOH (30:1) and then recrystalized withEtOH to give the title compound (4.5 g, 78%) as a white solid. ¹H NMR(DMSO-d₆) δ 11.46 (s, 1H), 9.22 (s, 2H), 8.66 (d, J=5.6 Hz, 1H), 8.31(s, 1H), 7.81 (d, J=5.6 Hz, 1H), 5.82 (q, J=7.2 Hz 1H), 5.12 (t, 1H),3.70 (m, 2H), 3.47 (s, 3H), 3.16 (s, 3H), 2.10 (m, 4H), 1.88 (d, J=7.2Hz, 3H). MH⁺ 545. diastereomeric excess (de): 99%*. *Chiral HPLC Methodcondition: Column: CHIRALPAK IB, 150*4.6 mm, 5 μm; Mobile Phase: A:Hexane (HPLC GRADE); B: EtOH (HPLC GRADE); Flow Rate: 0.8 mL/min;Gradient: 30% B for 25 min. Results: Retention time of the desireddiastereoisomer (S) is 14.16 min, the other isomer (R) is 9.66 min.

Example 3(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

The mother liquid after recrystallization from Example 2 in EtOH wasconcentrated. The residue was submitted for chiral preparative HPLCpurification to give(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamideas a white solid. ¹H NMR (DMSO-d₆) δ 11.48 (s, 1H), 9.25 (s, 2H), 8.69(d, J=5.6 Hz, 1H), 8.33 (s, 1H), 7.83 (d, J=5.6 Hz, 1H), 5.81 (q, J=7.2Hz 1H), 5.13 (t, 1H), 3.71 (m, 2H), 3.69 (s, 3H), 3.17 (s, 3H), 2.14 (m,4H), 1.88 (d, J=7.2 Hz, 3H). MH⁺ 545. de: 99%.* *Chiral HPLC Methodcondition: Column: CHIRALPAK IB, 150*4.6 mm, 5 μm; Mobile Phase: A:Hexane (HPLC GRADE); B: EtOH (HPLC GRADE); Flow Rate: 0.8 mL/min;Gradient: 30% B for 25 min.

Example 4(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((R)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

The title compound was prepared using the method of Example 2 to yield awhite solid. ¹H NMR (DMSO-d₆) δ 11.46 (s, 1H), 9.25 (s, 2H), 8.70 (d,J=5.6 Hz, 1H), 8.33 (s, 1H), 7.83 (d, J=5.6 Hz, 1H), 5.81 (q, J=7.2 Hz1H), 5.14 (t, 1H), 3.67 (m, 2H), 3.32 (s, 3H), 3.14 (s, 3H), 2.10 (m,4H), 1.86 (d, J=7.2 Hz, 3H). MH⁺ 545. de 98%* *Chiral HPLC Methodcondition: Column: CHIRALPAK IB, 150*4.6 mm, 5 μm; Mobile Phase: A:Hexane (HPLC GRADE); B: EtOH (HPLC GRADE); Flow Rate: 0.8 mL/min;Gradient: 30% B for 25 min.

Example 5(S)-2-(1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

To a mixture of(2S)-2-(1-methyl-2,6-dioxo-3,4,5,6-tetrahydro-1H-purin-7(2H)-yl)propanoicacid (90.4 mg, 0.38 mmol) and(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine(119 mg, 0.38 mmol) in DCM (2 mL) was added HOAT (104 mg, 0.76 mmol) atRT. The reaction was cooled to 0° C. Pyridine (91 mg, 1.15 mmol) wasadded dropwise followed by dropwise addition of DIC (97 mg, 0.77 mmol).The reaction was stirred at 20° C. for 16 h; then it was diluted withDCM (10 mL). The mixture was washed with saturated NH₄Cl and brine,dried over Na₂SO₄ and concentrated. The residue was purified bychromatography eluting with DCM:MeOH (30:1) to give the title product(83 g, 41%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.94 (s, 1H), 11.44(s, 1H), 9.25 (s, 2H), 8.71 (d, J=5.6 Hz, 1H), 8.23 (s, 1H), 7.84 (d,J=5.6 Hz, 1H), 5.78 (q, J=7.2 Hz 1H), 5.14 (t, 1H), 3.71 (m, 2H), 3.12(s, 3H), 2.20 (m, 4H), 1.84 (d, J=7.2 Hz, 3H). MH⁺ 531.

Example 6(S)-2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

To a mixture of(S)-2-(3-(difluoromethyl)-1-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (61 mg, 0.21 mmol) and(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine(310 mg, 0.21 mmol) in DCM (1 mL) was added HOAT (57 mg, 0.42 mmol) atRT. The mixture was cooled to 0° C. Pyridine (50 mg, 0.63 mmol) wasadded dropwise followed by dropwise addition of DIC (53 mg, 0.42 mmol).The reaction was stirred at 20° C. for 16 h, then diluted with DCM (10mL). The mixture was washed with saturated NH₄Cl and brine, dried overNa₂SO₄ and concentrated. The residue was purified by chromatographyeluting with DCM:MeOH (30:1) to give the product (45.6 mg, 37%) as awhite solid. ¹H NMR (DMSO-d₆) δ 11.49 (s, 1H), 9.25 (s, 2H), 8.70 (s,1H), 8.39 (s, 1H), 7.86 (t, J=5.6 Hz, 2H), 5.82 (q, J=8 Hz 1H), 5.14 (t,1H), 3.74 (m, 2H), 3.16 (s, 3H), 2.22 (m, 4H), 1.87 (d, J=8 Hz, 3H). MH⁺581.

Example 7N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide

A mixture of2-chloro-N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(50.0 mg, 0.1 mmol), K₂CO₃ (41.5 mg, 0.3 mmol),1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (26.5 mg, 0.1 mmol) and TBAI(5.6 mg, 0.01 mmol) in DMF (3 mL) was stirred at 90° C. overnight. Thereaction was cooled to RT and diluted with EA. The organic phase waswashed with water, saturated NH₄Cl solution and brine, dried overNa₂SO₄, and concentrated. The residue was purified by preparative TLC(DCM/MeOH=30:1) to giveN-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide(4.0 mg, 5.6%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.48 (s, 1H), 9.26(s, 2H), 8.73 (d, J=5.6 Hz, 1H), 8.08 (s, 1H), 7.83 (s, 1H), 5.36 (s,2H), 4.60 (t, J=12.2 Hz, 4H), 3.46 (s, 3H), 3.19 (s, 3H). MH⁺ 485.1.

Example 8N-(2′-(4,4-difluoropiperidin-1-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide

A mixture ofN-(2-chloropyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide(87.3 mg, 0.3 mmol), Pd(PPh₃)₄ (28.9 mg, 0.03 mmol),(2-(4,4-difluoropiperidin-1-yl)pyrimidin-5-yl)boronic acid (66.9 mg, 0.3mmol) and NaHCO₃ (21.0 mg, 0.3 mmol) in 1,4-dioxane (3 mL) and water(0.5 mL) was degassed with nitrogen and stirred at 90° C. overnight. Thereaction was cooled to RT and diluted with EA. The organic phase wasseparated, washed with water and brine, dried over Na₂SO₄ andconcentrated. The residue was purified by preparative TLC(DCM/MeOH=30:1) to giveN-(2′-(4,4-difluoropiperidin-1-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide(8.0 mg, 6.2%) as a white solid. ¹H-NMR (DMSO-d₆) δ 11.44 (s, 1H), 9.23(s, 2H), 8.71 (d, J=5.7 Hz, 1H), 8.08 (s, 1H), 7.81 (s, 1H), 5.36 (s,2H), 4.05-3.97 (m, 4H), 3.46 (s, 3H), 3.19 (s, 3H), 2.06 (dd, J=12.5,6.6 Hz, 4H). MH⁺ 513.1.

Example 9(S)—N-(2′-(3,3-difluoroazetidin-1-yl)-2,5′-bipyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamide

To a mixture of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (57 mg, 0.23 mmol) and2′-(3,3-difluoroazetidin-1-yl)-2,5′-bipyrimidin-4-amine (50 mg, 0.19mmol) in DCM (3 mL) was added HOAT (31 mg, 0.23 mmol) at RT. Thereaction was cooled to 0° C. then pyridine (30 mg, 0.38 mmol) was slowlyadded dropwise followed by dropwise addition of DIC (36 g, 0.29 mmol).The reaction was allowed to warm to RT and then warmed to 30° C. andstir 18 h. The reaction was extracted with water followed by saturatedNH₄Cl. The organic phase was dried over Na₂SO₄, and concentrated. Theresidue was purified by preparative HPLC (DCM/MeOH=30:1) to give(S)—N-(2′-(3,3-difluoroazetidin-1-yl)-2,5′-bipyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamide(10 mg, 9%) as white a solid. ¹H NMR (DMSO-d₆) δ 11.51 (s, 1H), 9.26 (s,2H), 872 (d, 1H), 8.35 (s, 1H), 7.86 (d, 1H), 5.81 (q, 1H), 4.60 (t,4H), 3.47 (s, 3H), 3.17 (s, 3H), 1.81 (d, 3H). MH⁺ 499.

Example 10N-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide

A mixture ofN-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-yl)-2-chloroacetamide (50.0 mg, 0.16 mmol), K₂CO₃ (44.3 mg, 0.32 mmol),1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (28.0 mg, 0.16 mmol) and TBAI(5.9 mg, 0.02 mmol) in DMF (3 mL) was stirred at 90° C. overnight. Thereaction was cooled to RT and diluted with EA. The organic phase wasextracted with water, saturated NH₄Cl and brine, dried over Na₂SO₄, andconcentrated. The residue was purified by preparative TLC(DCM/MeOH=25:1) to giveN-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide(2.0 mg, 2.9%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.41 (s, 1H), 9.17(s, 2H), 8.68 (d, J=5.7 Hz, 1H), 8.08 (s, 1H), 7.76 (s, 1H), 5.36 (s,2H), 3.87 (d, J=11.5 Hz, 2H), 3.59 (s, 2H), 3.51 (s, 3H), 3.19 (s, 3H),0.82 (m, 3H), 0.18 (s, 1H). MH⁺475.

Example 112-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)

A mixture of2-chloro-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(100.0 mg, 0.26 mmol), K₂CO₃ (53.7 mg, 0.39 mmol),1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (46.6 mg, 0.26 mmol) and TBAI(9.7 mg, 0.03 mmol) in DMF (3 mL) was stirred at 90° C. overnight. Thereaction was cooled to RT and diluted with EA. The organic phase waswashed with water, saturated NH₄Cl and brine, dried over Na₂SO₄, andconcentrated. The residue was purified by preparative TLC(DCM/MeOH=30:1) to give2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′bipyrimidin]-4-yl)acetamide (4.0 mg, 2.9%) as a white solid. ¹HNMR(DMSO-d₆) δ 11.42 (s, 1H), 9.22 (s, 2H), 8.70 (d, J=5.7 Hz, 1H), 8.08(s, 1H), 7.79 (s, 1H), 5.36 (s, 2H), 3.91 (dd, J=11.7, 8.1 Hz, 1H), 3.77(s, 1H), 3.72-3.61 (m, 2H), 3.46 (s, 3H), 3.19 (s, 3H), 2.35-2.29 (m,1H), 2.14 (dd, J=12.9, 7.3 Hz, 1H), 2.00 (d, J=7.9 Hz, 1H). MH⁺ 531.

Example 12N-(2′-(3,3-difluoroazetidin-1-yl)-[2,5′-bipyrimidin]-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamide

A mixture of2-chloro-N-(2-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-yl)acetamide(70 mg, 0.19 mmol), K₂CO₃ (51.9 mg, 0.38 mmol),1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (34.2 mg, 0.19 mmol) and TBAI(11.2 mg, 0.019 mmol) in DMF (2 mL) was stirred at 50° C. for 2 h. Thereaction was cooled to RT and diluted with EA. The organic phase waswashed with water and brine, dried over Na₂SO₄ and concentrated. Thecrude product which was triturated with MeOH, filtered, and dried togive the title product2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-7-yl)-N-(2-(2-(3-(trifluoromethyl)azetidin-1-yl)pyrimidin-5-yl)pyrimidin-4-yl)acetamide(7.0 mg, 5.6%) as a white solid. ¹H NMR (DMSO-d₆) δ 9.22 (s, 2H), 8.71(d, J=6.0 Hz, 1H), 8.08 (s, 1H), 7.81 (d, J=4.8 Hz, 1H), 7.83 (s, 1H),5.36 (s, 2H), 4.39-4.44 (m, 2H), 4.12-4.17 (m, 2H), 3.76-3.78 (m, 1H),3.45 (s, 3H), 3.21 (s, 3H). MH⁺ 517.

Example 13(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(3-(trifluoromethyl)azetidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

To a mixture of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (85 mg, 0.34 mmol) and2′-(3-(trifluoromethyl)azetidin-1-yl)-2,5′-bipyrimidin-4-amine (100 mg,0.34 mmol) in DCM (3 mL) was added HOAT (46 mg, 0.34 mmol) at RT. Thereaction was cooled to 0° C. Sequentially pyridine (54 mg, 0.68 mmol)and DIC (64 mg, 0.51 mmol) were slowly added dropwise. The reaction waswarmed to 30° C. and stirred 18 h. The reaction was washed with water (5mL), and saturated NH₄Cl (5 mL). The organic phase was dried over Na₂SO₄and concentrated. The residue was purified by chromatography elutingwith PE:EA (1:1) to afford(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(3-(trifluoromethyl)azetidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide(60 mg, 33.3%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.49 (s, 1H), 9.22(s, 2H), 8.70 (d, J=6.0 Hz, 1H), 8.34 (s, 1H), 7.84 (d, J=6.0 Hz, 1H),5.81 (q, J=8.0 Hz 1H), 4.42 (t, J=8.8 Hz, 2H), 4.13-4.17 (m, 2H),3.75-3.78 (m, 1H), 3.45 (s, 3H), 3.18 (s, 3H), 1.87 (d, J=8.8 Hz, 3H),MH⁺ 531.

Example 14(S)—N-(2′(4,4-difluoropiperidin-1-yl)-2,5′-bipyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamide

To a solution of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl) propanoicacid (50 mg, 0.2 mmol) and2′-(4,4-difluoropiperidin-1-yl)-2,5′-bipyrimidin-4-amine (58 mg, 0.2mmol) in DCM (3 mL) was added HOAT (27 mg, 0.2 mmol) at RT. The reactionwas cooled to 0° C. Sequentially pyridine (32 mg, 0.4 mmol) and then DIC(38 mg, 0.3 mmol) were added slowly dropwise. The reaction was warmed to30° C. for 18 h. The reaction was extracted with water (5 mL), andsaturated NH₄Cl (5 mL). The organic phase was dried over Na₂SO₄ andconcentrated. The residue was purified via preparative TLC eluting withPE: EtOAc (1:1) to afford(S)—N-(2′-(4,4-difluoropiperidin-1-yl)-2,5′-bipyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamide(15 mg, 14.3%) as a white solid. ¹H NMR (DMSO-d₆) δ 9.22 (s, 2H), 8.70(d, J=5.6 Hz, 1H), 8.34 (s, 1H), 7.83 (d, J=5.6 Hz, 1H), 5.81 (q, J=6.8Hz 1H), 4.02 (t, J=5.6 Hz, 4H), 3.54 (s, 3H), 3.21 (s, 3H), 2.01-2.10(m, 4H), 1.87 (d, J=6.8 Hz, 3H). MH⁺ 527.2.

Example 15(2S)—N-(2′-(3-azabicyclo[3.1.0]hexan-3-yl)-2,5′-bipyrimidin-4-yl)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamide

The title compound was prepared following the method of Example 14 with20.7% yield as a white solid. ¹H NMR (DMSO-d₆) δ 11.42 (s, 1H), 9.16 (s,2H), 8.66 (d, J=5.6 Hz, 1H), 8.33 (s, 1H), 7.79 (d, J=5.6 Hz, 1H), 5.81(q, J=7.2 Hz 1H), 3.86 (d, J=11.2 Hz, 2H), 3.57 (d, J=11.6 Hz, 2H), 3.41(s, 3H), 3.17 (s, 3H), 1.86 (d, J=7.6 Hz, 3H), 1.70 (t, J=3.6 Hz, 2H),0.75-0.80 (m, 1H), 0.15-0.18 (m, 1H). MH⁺ 489.

Example 16(2S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(3-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

The title compound was prepared following the method of Example 14 with39.1% yield as a white solid. ¹H NMR (DMSO-d₆) δ 11.46 (s, 1H), 9.22 (s,2H), 8.69 (d, J=5.6 Hz, 1H), 8.34 (s, 1H), 7.81 (d, J=5.6 Hz, 1H), 5.82(q, J=7.2 Hz, 1H), 3.88-3.94 (m, 1H), 3.69-3.78 (m, 1H), 3.62-3.67 (m,2H), 3.50 (s, 3H), 3.43-3.49 (m, 1H), 3.20 (s, 3H), 2.30-2.35 (m, 2H),2.12-2.17 (m, 1H), 1.87 (t, J=7.2 Hz, 3H). MH⁺ 545.2.

Example 17(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide

To a solution of 1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (208 mg, 1.16mmol) in DMF (8 mL) was added K₂CO₃ (320 mg, 2.32 mmol) and(S)-2-chloro-N-(2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide(400 mg, 1.16 mmol). The reaction was stirred at RT for 1 h; then pouredinto ice-water. The solid precipitate was collected and triturated withMeOH to afford(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide(220 mg, 45.5%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.38 (s, 1H), 9.17(s, 2H), 8.68 (d, J=5.6 Hz, 1H), 8.07 (s, 1H), 7.75 (s, 1H), 5.36 (s,2H), 5.12-5.14 (m, 1H), 4.67-4.71 (m, 1H), 3.45 (s, 3H), 3.19 (s, 3H),3.00 (t, J=12 Hz, 1H), 1.57-1.75 (m, 5H), 1.22-1.40 (m, 1H), 1.19 (d,J=8 Hz, 3H). MH⁺ 491.

Example 182-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2,2-dimethylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide

The title compound was prepared using the method of Example 17 with43.1% yield as a white solid. ¹H NMR (CDCl₃) δ 9.59 (s, 1H), 9.19 (s,2H), 8.60 (d, J=5.2 Hz, 1H), 7.77 (s, 2H), 5.18 (s, 2H), 3.77 (t, J=6.4Hz, 2H), 3.64 (s, 3H), 3.47 (s, 3H), 1.94-1.98 (m, 4H), 1.604 (s, 6H).MH⁺ 491.

Example 19(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)acetamide

The title compound was prepared using the method of Example 17 with46.7% yield as a white solid. ¹H NMR (DMSO-d₆) δ 11.38 (s, 1H), 9.18 (s,2H), 8.68 (d, J=5.2 Hz, 1H), 8.09 (s, 1H), 7.78 (s, 1H), 5.37 (s, 2H),4.32 (t, J=5.2 Hz, 1H), 3.54-3.68 (m, 2H), 3.47 (s, 3H), 3.20 (s, 3H),1.71-2.11 (m, 4H), 1.24 (d, J=6.4 Hz, 3H). MH⁺ 477.

Example 202-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

To a solution of 1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (312 mg, 1.73mmol) in DMF (8 mL) was added K₂CO₃ (477 mg, 3.46 mmol),2-chloro-N-(2′-((S)-2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide(600 mg, 1.73 mmol). The reaction was stirred at RT for 1 h; then pouredinto ice water. The solid precipitate was collected and washed with MeOHto afford 2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide(130 mg, 16%) as a white solid. ¹H NMR (DMSO-d₆) δ 11.43 (s, 1H), 9.18(s, 2H), 8.67 (d, J=5.6 Hz, 1H), 8.34 (s, 1H), 7.78 (d, J=5.6 Hz, 1H),5.82 (d, J=7.2 Hz, 1H), 4.31 (t, J=5.2 Hz, 1H), 3.53-3.68 (m, 2H), 3.46(s, 3H), 3.19 (s, 3H), 1.71-2.19 (m, 7H), 1.23 (d, J=6.4 Hz, 3H). MH⁺491.

Example 21(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

The diastereomeric product mixture from Example 20 was separated bysupercritical fluid chromatography with a chiral column* to obtain(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamideas colorless liquid (retention time 6.0 min). MH⁺ 491. * The chiral HPLCseparation conditions 2.1×25.0 cm ChiralPak IC from Chiral Technologies(West Chester, Pa.), with 50% supercritical carbon dioxide and 50% of a2:1:1 mixture of DCM:Hexane:Isopropanol at a flow rate of 80 mL/min.

Example 22(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

The diastereomeric product mixture from Example 20 was separated bysupercritical fluid chromatography with a chiral column* to obtain(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamideas colorless liquid (retention time 4.8 min). MH⁺ 491. * The chiral HPLCseparation conditions 2.1×25.0 cm ChiralPak IC from Chiral Technologies(West Chester, Pa.), with 50% supercritical carbon dioxide and 50% of a2:1:1 mixture of DCM:Hexane:Isopropanol at a flow rate of 80 mL/min.

Example 232-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-methylpiperidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

The title product was prepared using the methods of Preparation 26 andExample 20 with 28.3% yield as a white solid. ¹H NMR (CDCl₃) δ 9.73 (d,J=6.4 Hz, 1H), 9.17 (s, 2H), 8.56 (d, J=5.6 Hz, 1H), 7.86 (d, J=7.6 Hz,1H) 7.79 (d, J=4 Hz, 1H), 5.76 (d, J=6.4 Hz, 1H), 4.76 (d, J=12.4 Hz,1H), 3.60 (s, 3H), 3.47 (s, 3H), 3.02 (t, J=12 Hz, 1H), 1.90 (d, J=7.2Hz, 3H), 1.48-1.78 (m, 6H), 1.23 (d, J=6.4 Hz, 3H). MH⁺ 505.

Example 24(S)-2-(3-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide

(S)-2′-(2-methylpyrrolidin-1-yl)-2,5′-bipyrimidin-4-amine (249 mg, 0.971mmol), 2-(3-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetic acid(436 mg, 1.943 mmol) and EDC (745 mg, 3.89 mmol) were added to pyridine(2.43 mL). The reaction was stirred 2 days at RT. The reaction wasconcentrated and the residue was purified by chromatography to afford(S)-2-(3-methyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide(50 mg, 11%) as colorless liquid. MH⁺ 463.

Example 25N-{2-[2-((2S)-2-methylpyrrolidinyl)pyrimidin-5-yl]pyrimidin-4-yl}-2-(1-methyl-3-methyl-d3-2,6-dioxo(1,3,7-trihydropurin-7-yl))acetamide

A mixture of2-(1-methyl-3-methyl-d3-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)aceticacid (0.200 g, 0.829 mmol),(S)-2′-(2-methylpyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine (0.213 g,0.829 mmol) and EDC (0.318 g, 1.658 mmol) was dissolved in pyridine(4.15 mL) at RT. The reaction was stirred overnight and then dilutedwith water. The reaction was extracted three times with EA. The organicphase was dried over MgSO₄ and concentrated. The residue was purified bychromatography to affordN-{2-[2-((2S)-2-methylpyrrolidinyl)pyrimidin-5-yl]pyrimidin-4-yl}-2-(1-methyl-3-methyl-d3-2,6-dioxo(1,3,7-trihydropurin-7-yl))acetamide(66.1 mg 17%) as colorless liquid. MH⁺ 480.

Example 26((2S)—N-(2-(2-(3-azabicyclo[3.1.0]hexan-3-yl)pyramidin-5-yl)thiazol-4-yl)-2-(3-methyl-2,6-dioxo-1-(2-oxobutyl)-2,3-dihydro-1H-purin-7(6H)-yl)propanamide

The title compound was prepared using the method of Example 14 in 8%yield as a white solid. ¹H NMR (DMSO_(d6)) δ 11.47 (s, 1H), 9.20 (s,2H), 8.69 (d, J=5.1 Hz, 1H), 8.35 (s, 1H), 7.82 (d, J=5.1 Hz, 1H), 5.83(s, 1H), 4.01 (d, J=12.0 Hz, 2H), 3.87 (d, J=11.0 Hz, 2H), 3.46 (s, 3H),3.18 (s, 3H), 2.71 (d, J=11.5 Hz, 2H), 1.87 (d, J=6.6 Hz, 3H). MH⁺ 525.

Example 27(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)acetamide

To a mixture of2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetyl chloride(250 mg, 0.97 mmol) and(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine(332 mg, 1.07 mmol) in THF (10 mL) was added DIPEA (0.509 mL, 2.92 mmol)at 0° C. The reaction was stirred at RT for 18 h, then refluxed for 24h. The mixture was cooled to RT, diluted with water (75 mL) andextracted with EA (50 mL×3). The combined organic layers were dried overMgSO₄, and concentrated. The residue was purified by preparative TLC(eluting with 100% EA) to give the title compound (17 mg, 3.3%) as awhite solid. ¹H NMR (CDCl₃) δ 9.6 (brd s, 1H), 9.26 (s, 2H), 8.61 (d,J=4 Hz, 1H), 7.96-7.8 (m, 1H), 7.75 (s, 1H), 5.22-5.06 (m, 3H), 5.12 (t,1H), 3.89-3.75 (m, 2H), 3.62 (s, 3H), 3.46 (s, 3H), 2.35-2.22 (m, 2H),2.18-2.04 (m, 2H). MH⁺ 531.

Example 28(R)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-aR)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamide

The title compound was prepared using the method of Example 2 to yieldthe title compound as a white solid. ¹H NMR (DMSO-d₆) δ 11.46 (s, 1H),9.22 (s, 2H), 8.66 (d, J=5.6 Hz, 1H), 8.31 (s, 1H), 7.81 (d, J=5.6 Hz,1H), 5.82 (q, J=7.2 Hz 1H), 5.12 (t, 1H), 3.70 (m, 2H), 3.47 (s, 3H),3.16 (s, 3H), 2.10 (m, 4H), 1.88 (d, J=7.2 Hz, 3H). MH⁺ 545

Example 29 Process Scale Synthesis of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-yl)propanamideStep 1 (R)-Methyl 2-(((trifluoromethyl)sulfonyl)oxy)propanoate

A 50 L reactor under a nitrogen atmosphere was charged with methylenechloride (30 L) and agitated. (R)-methyl lactate (1.44 kg, 13.83 mol)was added, followed by 2,6-lutidine (1.56 kg, 14.56 mol). The stirredmixture was cooled to −5 to 5° C. using a dry-ice/acetone bath. Thereactor was carefully charged with trifluoromethane sulfonic anhydride(3.9 kg, 13.83 mol) using a peristaltic pump while maintaining theinternal temperature between −5 and 5° C. This addition required morethan 1 h. After addition was complete, the reaction was stirred 1 h morewhile maintaining the temperature between 0 and 5° C. The reaction wascarefully quenched with deionized water (10 L) and vigorous stirring wascontinued 1 min longer. Stirring was stopped and the phases were allowedto separate. The bottom (methylene chloride) layer containing theproduct, was transferred to a holding container while the reactor wascleaned successively with acetone (2×10 L) and then methylene chloride(2×10 L). The intermediate triflate was used directly withoutpurification.

Step 2 Methyl(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoate

The methylene chloride product solution of R-Methyl2-(((triflluoromethyl)sulfonyl)-oxy)propanoate was charged back to theclean 50 L reactor and the mixture was placed under a nitrogenatmosphere. The mixture was cooled with stirring to 0 to 5° C. with adry ice/acetone bath. During cooling theophylline (2.0 kg, 11.1 mol) wascharged to the reactor. 1,1,3,3-Tetramethylguanidine (1.34 kg, 11.66mol) was slowly added to the reactor via peristaltic pump whilemaintaining the internal temperature below 10° C. After the addition wascomplete, the reaction was stirred for at least 1 h while maintainingthe reaction temperature at 0 to 10° C. An aliquot taken after 30 minwas tested by HPLC and confirmed the reaction was complete. Ice-cold0.2N HCl (10 L) was added to the reactor to quench the reaction. Themixture was stirred vigorously for 1-2 min. Stirring was stopped and thephases were allowed to separate. The bottom methylene chloride productlayer was transferred to a holding container. The upper aqueous layerwas removed and discarded. The bottom methylene chloride layer was addedback to the reactor and it was extracted with 5% aqueous NaHCO₃ (10 L)with vigorous stirring for 1-2 min. Stirring was stopped and the phaseswere allowed to separate. The bottom product layer was transferred to aholding container. The upper aqueous layer was removed and discarded.The bottom methylene chloride layer was added back to the reactor.Deionized water (10 L) was added to the reactor. The mixture was stirredvigorously for 1 min. Stirring was stopped and the phases were allowedto separate. The bottom product layer was transferred to a holdingcontainer. The upper aqueous layer was removed and discarded. Themethylene chloride product-containing solution was transferred to arotary evaporator and concentrated under vacuum (bath temperature 30-40°C.) until most of the methylene chloride distilled leaving crude methyl(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoateas a dark viscous syrup. HPLC analysis of a sample confirmed the productand its purity.

Step 3(S)-2-(1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid

Methyl(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoate(7.16 kg, crude syrup from two batches from Steps 1 and 2) wastransferred to a rotary evaporator bulb. Vacuum was applied and the bulbwas rotated with a bath temperature of 30-40° C. until no more methylenechloride was distilled. Separately, a solution of 3N aqueous HCl (32 L,4.5 equiv, based on 4 kg of theophylline) was prepared. The residue fromthe rotary evaporator bulb was transferred to a 50 L reactor. The bulbwas rinsed with small portions of 3N HCl to remove all crude ester andtransferred to the reactor, and the remaining 3N HCl was charged to thereactor. The reaction mixture was heated at 70-75° C. for at least 16 h.The reaction status at 16 h was checked by HPLC analysis of a smallaliquot, and was deemed complete when the amount of ester was less than10% compared to the acid product. The mixture was allowed to cool toroom temperature with stirring for at least 16 h. The product wascollected on a Buchner funnel and the solids were washed with ice-colddeionized water (2×2 L). The solids were dried on the vacuum funnelovernight until the mixture became a free-flowing solid (2.95 Kg). Thecrude product was 93.8% pure by HPLC analysis.

A portion of the crude(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (1 kg) was charged into a 22 L reactor. Deionized water (9 L, 9volumes) was added to the reactor and stirring was started. The slurrywas heated to 95° C. and held at that temperature until all the solidsdissolved. A slurry of 40 g (4% by wt.) of Norite® activated carbon in250 mL of deionized water was added to the hot mixture and stirring wascontinued at 90-95° C. for 1 h. The hot mixture was carefullytransferred from the 22 L vessel through a filter funnel containing aglass microfiber filter into a clean 50 L reactor. This process wasrepeating two more times with 1 kg of the crude acid, each timefiltering into the same 50 L reactor. The reactor was allowed to cool tobelow 30° C. with stirring. The solids were filtered and the product waswashed with ice-cold deionized water (2×2 L). The product was dried atleast 12 h on the filter funnel, then it was transferred to a vacuumoven and dried to a constant weight to afford(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (2.25 kg). The purified product was 99.31% pure by HPLC and had anenantiomeric purity of 100% by chiral HPLC. The overall yield of pure(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid based on the theophylline starting material was 42.4%.

Step 4 (S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine

To a reaction vessel equipped with mechanical stirring, refluxcondenser, nitrogen inlet, thermocouple, and an external heating mantlewas charged with (S)-2-trifluoromethylpyrrolidine (1598 g, 11.49 mol),5-bromo-2-chloropyrimidine (2000 g, 10.34 mol) and N,N-dimethylacetamide(9 L). The stirred mixture was warmed to 50° C. When the mixture becamea solution, diisopropylethylamine (1633 g, 12.64 mol) was added and thereaction temperature was increased to 120° C. The reaction is stirredfor 24-48 h until the reaction is complete by HPLC. The reaction iscooled to no less than 70° C. and the contents are transferred to asecond stirred vessel containing water (90 L). This mixture was stirredand allowed to cool to 20° C., then further cooled to 5 to 10° C. andheld at this temperature for 2 h. The solid product is collected byfiltration and washed with cold water (3×5 L) The product was driedunder vacuum at 50° C. to constant weight to afford(S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine (2898 g,94.6%) as a light brown solid that was ˜99% pure by HPLC.

Step 5(S)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine

A reaction vessel equipped with mechanical stirring, reflux condenser,nitrogen inlet, thermocouple, and an external heating mantle was chargedwith dioxane (8 L) and gentle stirring was initiated. The reaction wascharged with(S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine (1600 g,5.40 mol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(2058 g, 8.11 mol), and potassium acetate (1059 g, 10.81 mol).Additional dioxane (17 L) was added and nitrogen gas was bubbled throughthe mixture. Bis-(triphenylphosphine) palladium chloride catalyst (113.7g, 0.161 mol) was added to the reaction. The reaction was heated withnitrogen still bubbling through the mixture. When the reactiontemperature reached 50° C., nitrogen was no longer bubbled through themixture. However a nitrogen atmosphere was maintained, venting throughthe condenser. The reaction temperature was increased to 95 to 100° C.and maintained at this temperature until HPLC analysis indicated thereaction was complete, after about 16-24 h. The reaction was cooled tono less than 60° C., and transferred via peristaltic pump into a reactorcontaining 38 volumes of water. The transfer line was rinsed with 0.25to 1.50 vol of dioxane. Additional water was added to the reactor whenappropriate to facilitate product crystallization. The mixture wascooled to 10±5° C. and held for at least 1 hour. The product wascollected in a Buchner funnel, washed with cold water (3×2 volumes), anddried under vacuum at 50-60° C. until a constant weight was achieved.(S)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidinewas obtained as a light brown solid, 1964 g. This solid contained 12.7%water and was approximately 96% purity as determined by HPLC. The yieldwas estimated to be 1715 g, 91.4%. The material was suitable for furtherreaction without removal of residual water.

Step 6(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine

The wet(S)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine(1964 g) from Step 5 was assayed for water content, and thestoichiometry was adjusted accordingly (1715 g, 5.0 mol). Dioxane (16 L)was added to a reaction vessel equipped with a heating mantle,thermocouple controller, nitrogen inlet, mechanical stirrer and refluxcondenser. Compounds(S)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine(corrected to 1715 g, 5.0 mol), 4-amino-2-chloropyrimidine (648 g, 5mol) and sodium carbonate (962 g, 9.08 mol) were charged to the reactionvessel. Additional dioxane (8 L) is added. Nitrogen gas was bubbledthrough the solution for about 30-60 min., venting through thecondenser. Tetrakis (triphenylphosphine) palladium (230 g 0.2 mol) wasadded, and the residual catalyst was rinsed into the reaction vesselwith dioxane (1 L). Heating was started, and nitrogen bubbling wascontinued until the mixture reached about 50° C. At this time thenitrogen tube was retracted above the surface of the solution, butnitrogen the nitrogen atmosphere was maintained, venting through thecondenser. The temperature was increased to 85-90° C. and maintaineduntil the reaction was complete (1-4 h) as determined by HPLC. Thereaction was cooled to no less than 60n ° C., and water (18 L) was addedwhile maintaining temperature. The reaction mixture was filtered hotthrough GF-B glass fiber paper into a filter bottle. The filtrate wastransferred while warm into a reactor, rinsing with 1:1 dioxane/water(0.5 to 3 L) as necessary, with the reactor jacket temperature set to45° C. Water (36 L) was then added to the reactor, and the temperaturewas maintained during addition. The mixture was slowly cooled to 5±5°C., and additional water was added to the reactor as needed to maximizecrystallization. The temperature was held for at 5±5° C. for at least 2hours, after which the product was collected in a Buchner funnel andwashed with cold water (3×3.5 L). The product was dried under vacuum at50-60° C. until a constant weight was achieved to give(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine(1073 g, 70%) as an off-white solid.

A portion of this crude product (754 g, 2.43 mol) was slurried with 3NHCl (15 L) and filtered to remove impurities. The acidic solution wasextracted with MTBE (4 L)) and heptane (4 L). These organic extractswere discarded to waste. The acidic solution was basified with 50%sodium hydroxide to pH 9-10. The mixture was cooled, and the precipitatecollected by filtration. The solid was washed with cold water and driedunder vacuum to yield an off-white solid, 695 g, 92% recovery. Residualpalladium in this material was 782 ppm. This product was recrystallizedfrom 50% aqueous acetonitrile (8 L). The mixture was cooled, and theproduct collected by filtration, rinsed with cold solvent and driedunder vacuum to give 401.7 g (53% of the initial 754 g) of off-whitesolid, now with 181 ppm residual palladium. This off-white solid wasdissolved in THF and treated with palladium scavenger SillCycle®SiliaMetS® Thiol resin (25 g). The solvent was removed to give(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amineas a white solid (390 g, 97% recovery) that was greater than 97% pure byHPLC and with less than 10 ppm residual palladium.

Step 7(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide

To a 100 L reactor with agitator, nitrogen inlet, and condenser wasadded dichloromethane (20 L),(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanoicacid (1.0 kg, 3.96 mol) and N,N-dimethylformamide (14.5 mL, 0.2 mol).Additional dichloromethane (10 L) was added and the stirred mixture waschilled to 10-15° C. Oxalyl chloride (1.51 kg, 11.9 mol) was slowlyadded while maintaining the temperature below 25° C. The reaction wasstirred 30-60 min at 25° C. The solvent was distilled from the reactionunder vacuum and with a nitrogen bleed and the reactor jackettemperature increased to 35° C. as necessary. Additional dichloromethane(20 L) was added and the solvent was again distilled from the reaction.The addition and distillation of dichloromethane was repeated.Tetrahydrofuran (10 L) was added and this yielded a white to beigeslurry. To the reaction was added(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine(1.07 kg, 3.45 mol) and the addition vessel was rinsed withtetrahydrofuran (1.5 L) into the reaction mixture. The reaction wascooled to ≦0° C. and 2,6-lutidine (0.964 L, 8.28 mol) was added,maintaining the reaction temperature below 5° C. The reaction wasstirred at about 0° C. until it was considered complete by HPLC (about2% of(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amineremaining). After about 14 h, the reaction was carefully quenched with0.2 N HCl (40 L) and water (20 L) while maintaining the reactiontemperature below 15° C. The solid product was collected and washed withdeionized water twice. The solids were dried to constant weight undervacuum at 50-60° C. to afford 1,473 g (78%) of(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoromethyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamide.This product material was combined with 1,435 g of product from asimilar reaction and recrystallized from 2% aqueous ethanol (86.8 L) toyield 2,250 g of purified(S)-2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)-N-(2′-((S)-2-(trifluoro-methyl)pyrrolidin-1-yl)-2,5′-bipyrimidin-4-yl)propanamideas an off-white solid.

Example 30 Telescoped synthesis of(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-aminefrom (S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine

To a reaction vessel charged with dioxane (17.25 L) is added(S)-5-bromo-2-(2-(trifluoromethyl)pyrrolidin-1-yl)pyrimidine (1500 g,5.066 mol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(1937 g, 7.628 mol), and potassium acetate (998 g, 10.17 mol).Additional dioxane (6.25 L) was added and nitrogen gas was bubbledthrough the mixture with gentle stirring for 30 to 60 min.Bis-(triphenylphosphine) palladium chloride catalyst (107 g, 0.152 mol)was added with a dioxane rinse (0.5 L), and the reaction was heated to50° C. At this point, nitrogen was no longer bubbled through themixture, although a nitrogen atmosphere was maintained, venting throughthe condenser. The reaction temperature was further increased to 95 to100° C. and maintained at this temperature until the reaction wascomplete as indicated by HPLC analysis (about 24 h).

The reaction was then cooled to 60° C. and 4-amino-2-chloropyrimidine(623 g, 4.81 mol), sodium carbonate (975 g, 9.2 mol), and water (7.5 L)were added. Nitrogen gas was bubbled through the solution for about30-60 min, venting through the condenser. Tetrakis (triphenylphosphine)palladium (129 g 0.11 mol) was added, and the residual catalyst wasrinsed into the reaction vessel with dioxane (0.5 L). Heating wasrestarted, and nitrogen bubbling was continued until the mixture reachedabout 50° C. At this time the nitrogen tube was retracted above thesurface of the solution, but the nitrogen atmosphere was maintained,venting through the condenser. The temperature was increased to 85-90°C. and maintained until the reaction was complete (1-24 h) as determinedby HPLC. After cooling to no less than 60° C., water (15 L) was addedwhile maintaining the temperature and the reaction mixture was filteredthrough GF-B glass fiber paper into a filter bottle. The filtrate wastransferred while warm into a reactor, rinsing with 1:1 dioxane/water(0.5 to 3 L) as necessary, with the reactor jacket temperature set to45° C. Water (42 L) was added to the reactor, and the mixture was slowlycooled to 5±5° C. Additional water was added to the reactor as needed tomaximize crystallization, and the temperature was held at 5±5° C. for atleast 2 hours. The product was then collected in a Buchner funnel,washed with cold water (3×3.5 L), then dried under vacuum at 50-60° C.until a constant weight was achieved to give(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-amine(1266 g, 70%) as an off-white solid. This product was purified asdescribed in Example 29 (Step 7) to produce(S)-2′-(2-(trifluoromethyl)pyrrolidin-1-yl)-[2,5′-bipyrimidin]-4-aminewith similar purity as described.

Characterization of Compounds of the Invention

Compounds of the invention are referred to herein by their respectiveexample number. For example the compound produced by the methoddescribed in Example 1 may be referred to as “Compound of Example 1”,“Example 1”, or “Compound 1.” All three names may be used hereininterchangeably.

Example 31 Characterization of the Solid Crystalline Forms Obtained fromSlurry Treatment of Compounds of Formula (I)

Certain compounds of the invention produced solvate crystalline formsafter slurry treatment in a solvent or combination of solvents (e.g.,water, ethanol, or a combination thereof). For example, Compound 2produced a solvate crystalline form when slurried in ethanol or aqueousethanol mixtures containing up to 3% water at room temperature, referredherein as Form A. The solid crystalline product obtained from the slurrywas indexed using X-ray powder diffraction (XRPD) to define the unitcell (FIG. 1). The observed XPRD peaks are listed below in Table 1.

TABLE 1 Observed X-ray powder diffraction peaks from a solid crystallineform of Compound 2 (Form A) obtained from an ethanol slurry Intensity°2θ d space (Å) (%)  5.71 ± 0.20 15.452 ± 0.540  16  6.13 ± 0.20 14.396± 0.469  27  7.67 ± 0.20 11.515 ± 0.300  86  8.75 ± 0.20 10.094 ± 0.230 15  9.05 ± 0.20 9.763 ± 0.215 8  9.58 ± 0.20 9.229 ± 0.192 33 10.62 ±0.20 8.325 ± 0.156 23 11.73 ± 0.20 7.536 ± 0.128 10 12.31 ± 0.20 7.183 ±0.116 13 12.52 ± 0.20 7.067 ± 0.112 56 12.91 ± 0.20 6.851 ± 0.106 913.12 ± 0.20 6.744 ± 0.102 7 13.49 ± 0.20 6.559 ± 0.097 100 13.81 ± 0.206.408 ± 0.092 14 14.12 ± 0.20 6.268 ± 0.088 36 14.95 ± 0.20 5.921 ±0.079 17 15.40 ± 0.20 5.747 ± 0.074 19 16.10 ± 0.20 5.501 ± 0.068 1916.44 ± 0.20 5.388 ± 0.065 13 16.60 ± 0.20 5.336 ± 0.064 19 17.36 ± 0.205.105 ± 0.058 8 17.59 ± 0.20 5.038 ± 0.057 12 17.85 ± 0.20 4.964 ± 0.05512 18.90 ± 0.20 4.692 ± 0.049 28 19.31 ± 0.20 4.593 ± 0.047 55 19.69 ±0.20 4.505 ± 0.045 13 19.88 ± 0.20 4.463 ± 0.044 14 20.33 ± 0.20 4.366 ±0.043 15 20.55 ± 0.20 4.318 ± 0.042 13 20.86 ± 0.20 4.255 ± 0.040 2420.97 ± 0.20 4.232 ± 0.040 21 21.60 ± 0.20 4.111 ± 0.038 13 22.17 ± 0.204.007 ± 0.036 19 22.72 ± 0.20 3.911 ± 0.034 15 23.04 ± 0.20 3.857 ±0.033 14 23.18 ± 0.20 3.834 ± 0.033 33 23.32 ± 0.20 3.811 ± 0.032 1523.69 ± 0.20 3.752 ± 0.031 20 24.21 ± 0.20 3.673 ± 0.030 16 24.39 ± 0.203.646 ± 0.029 18 24.51 ± 0.20 3.629 ± 0.029 36 25.15 ± 0.20 3.538 ±0.028 17 25.92 ± 0.20 3.434 ± 0.026 18 26.77 ± 0.20 3.327 ± 0.024 727.55 ± 0.20 3.235 ± 0.023 8 28.29 ± 0.20 3.152 ± 0.022 10 28.85 ± 0.203.092 ± 0.021 7 29.13 ± 0.20 3.063 ± 0.021 25 29.42 ± 0.20 3.033 ± 0.0208 30.16 ± 0.20 2.961 ± 0.019 12

Drying of crystals of compound of Formula (I) obtained from slurrytreatment was capable of producing alternate polymorphs. For example,vacuum treatment (−80° C. for one day) of crystals of Compound 2obtained from slurry treatment in 97% ethanol/3% water produced astable, anhydrous solid crystalline form, referred to herein as Form B.This resulting crystalline form was characterized by using a variety ofmethods, including XRPD, polarized light microscopy, differentialscanning calorimetry (DSC), thermal gravimetric analysis (TGA), anddynamic vapor sorption (DVS) with post-DVS XRPD.

FIG. 2 shows the XRPD pattern of a sample of the anhydrous solidcrystalline form of Compound 2, and the observed peaks are listed belowin Table 2. The successful indexing of this sample indicates that it iscomposed of a single crystalline phase. After storage of this solidcrystalline form at ambient conditions for three months, the sample wassubjected to XRPD analysis again. The resulting XRPD pattern matches theoriginal indexed pattern shown in FIG. 2.

TABLE 2 Observed X-ray powder diffraction peaks from an anhydrous solidcrystalline form of Compound 2 (Form B) from an ethanol slurry Intensity°2θ d space (Å) (%)  5.70 ± 0.20 15.496 ± 0.543  35  7.86 ± 0.20 11.242± 0.286  11  8.20 ± 0.20 10.769 ± 0.262  24  9.41 ± 0.20 9.394 ± 0.19914  9.78 ± 0.20 9.033 ± 0.184 55 10.21 ± 0.20 8.654 ± 0.169 8 10.98 ±0.20 8.049 ± 0.146 25 11.45 ± 0.20 7.721 ± 0.134 17 11.75 ± 0.20 7.528 ±0.128 21 12.54 ± 0.20 7.053 ± 0.112 25 12.98 ± 0.20 6.813 ± 0.105 10013.71 ± 0.20 6.453 ± 0.094 10 14.31 ± 0.20 6.184 ± 0.086 29 14.80 ± 0.205.982 ± 0.080 22 15.72 ± 0.20 5.632 ± 0.071 21 15.97 ± 0.20 5.544 ±0.069 10 16.40 ± 0.20 5.400 ± 0.065 13 16.90 ± 0.20 5.243 ± 0.062 1317.32 ± 0.20 5.116 ± 0.059 15 17.65 ± 0.20 5.020 ± 0.056 18 17.88 ± 0.204.957 ± 0.055 16 18.10 ± 0.20 4.898 ± 0.054 14 18.84 ± 0.20 4.707 ±0.050 12 19.20 ± 0.20 4.619 ± 0.048 61 19.67 ± 0.20 4.509 ± 0.045 6520.58 ± 0.20 4.312 ± 0.041 10 21.16 ± 0.20 4.196 ± 0.039 10 22.03 ± 0.204.031 ± 0.036 8 23.04 ± 0.20 3.857 ± 0.033 14 23.42 ± 0.20 3.795 ± 0.03219 24.49 ± 0.20 3.633 ± 0.029 31 25.00 ± 0.20 3.558 ± 0.028 11 26.16 ±0.20 3.403 ± 0.026 10 27.09 ± 0.20 3.289 ± 0.024 13 28.04 ± 0.20 3.180 ±0.022 9 28.83 ± 0.20 3.094 ± 0.021 17 29.74 ± 0.20 3.002 ± 0.020 2430.01 ± 0.20 2.975 ± 0.019 19

Differential scanning calorimetry (DSC) and thermal gravimetric analysis(TGA) were also carried out on the anhydrous solid crystalline form ofCompound 2 (Form B); the resulting data are shown in FIG. 3 and FIG. 4.The DSC shows a minor broad endotherm with a peak maximum at 48.5° C.which is often indicative of a volatilization event; however there is nocorresponding weight loss event in the TGA. The broad endotherm at 48.5°C. may indicate the presence of absorbed water in the sample duringstorage which is consistent with the moisture sorption (DVS) data. TheDSC also shows a broad endotherm with a calculated onset of 185.4° C.that likely corresponds to a melting event and coincides with a minorTGA weight loss of 0.1% weight indicating that the sample may contain asmall amount of an unidentified volatile component.

Dynamic vapor sorption (DVS) analysis of the anhydrous solid crystallineform of Compound 2 (Form B) was also carried out. The resulting isothermplot is depicted in FIG. 5, and shows a 0.2% weight loss on equilibriumat 5% relative humidity (RH), followed by a reversibleadsorption/desorption of 2.7% weight with negligible hysteresis. Basedon this behavior, Form B appears to be a variable hydrate, in which thewater content will depend on the ambient relative humidity. Takentogether, the XRPD, DSC, TGA, and DVS data are all consistent with FormB being a crystalline, variable hydrate material that becomes anhydrousupon drying.

Crystals of Compound 2 obtained from ethanol or aqueous ethanol mixturesform long thin needles. XRPD patterns of such crystalline materials areoften complicated by the phenomenon of preferred orientation, resultingfrom the crystals predominantly aligning in two dimensions. Thus theXRPD patterns of compounds of Formula (I), e.g., Compound 2, obtainedfrom recrystallized samples look different from those obtained from theslurry experiments described above. It is known that particle sizereduction can reduce the magnitude of preferred orientation artifacts.FIG. 6 illustrates examples of XRPD patterns of the solid crystallineform of Compound 2 (Form B) recrystallized from ethanol and dried,before (light gray trace) and after micronization (dark gray tracetrace) to a d₉₀ value of less than 10 microns.

Example 32 Measuring Kinetic Solubilities of the Compounds of Formula(I)

The solubilities of the compounds of Formula (I) were tested using theprocedure outlined in Kerns, E. H., J Pharm Sci (2001) 90:1838-1858,incorporated herein by reference and described below. Data forsolubility was obtained by this method for compounds of Formula (I) andincluded in Table 3. The chromatographic data was performed by HPLCusing an Xbridge Shield RP18 column with the following columndimensions: 4.6×30 mm, 3.5 μm. The mobile phase consisted of deionizedwater (MPA) with trifluoroacetic acid added in at 0.1% (v/v) (MPC) andHPLC-grade acetonitrile (MPB). The mobile phase flow rate was 2.5 mL/minwith the column and sampling operating at ambient temperature. UVdetection was set to 280 nm. For all samples used for solubilitydetermination, the mobile phase gradient used is as shown in Table 4.

TABLE 3 Exemplary solubilities for selected compounds of Formula (I): pH4.0 pH 7.4 pH 9.0 solubility solubility solubility Compound (μg/mL)(μg/mL) (μg/mL) 1 4 3 2 2 85 71 69 17 11 12 9 23 20 31 14 25 45 29 11 2250 89 70 18 51 41 17 21 34 20 10.5 19 6 10 3.5

Samples for analysis of the compounds of Formula (I) were prepared at a% v/v=1/19 (i.e., 10 μL of the stock solution into 190 μL of buffer) byspiking stock solutions of the compounds of Formula (I) into bufferedsolutions. Three buffered solution systems were prepared: pH 4.0prepared from 50 mM sodium acetate in a 5% dextrose in water solution,pH 7.4 prepared from 75 mM sodium phosphate in a 1:1 ratio of sterilizedwater for injection to a 5% dextrose in water solution, and pH 9.0prepared from 50 mM sodium bicarbonate in a 1:2 ratio of sterilizedwater for injection to a 5% dextrose in water solution. The samples wereincubated on a microplate shaker at 300 rpm for 24 hours at ambienttemperature. Following incubation, the samples were centrifuged for fiveminutes at 13 k rpm at ambient temperature. The resulting supernantantwas extracted for HPLC analysis.

TABLE 4 Mobile phase gradient used for solubility determination Time(min) % MPA % MPB % MPC 0.00 70 20 10 1.08 0 90 10 1.20 0 90 10 1.21 7020 10 1.50 70 20 10

For example, compounds of the invention may allow acceptable levels ofdrug to reach therapeutic targets.

Solubility of a micronized formulation of Compound 2 was furtherevaluated using McIlvain's citrate-phosphate buffer recipes (0.2MNa₂HPO₄ and 0.1M citric acid) from pH 2.2 to 8.64. Samples were agitatedfor 30 hours and sampled, centrifuged, and analyzed by UPLC. The highestsolubility was seen at pH 8.6 and 3.1 while the effect of pH was narrowranging from 0.2 to 1.3 mg/ml. Results are shown in FIG. 10.

Solubility was also determined in a fasted-state simulated intestinalfluid (FaSSIF) assay. Briefly, Compound 2 was added to the FaSSIF medium(bile salts, NaOH (0.420 g), NaH2PO4 (3.438 g), NaCl (6.186 g), pH to6.5, at 25° C., and brought to a 1 L volume) agitated for 30 hours andsampled, centrifuged, and analyzed by UPLC. Solubility in the FaSSIFmodel was determined to be 1.19 mg/ml.

Solubility was further assessed in simulated gastrointestinal fluid(SGF) (HCl 0.1N at 25° C.). Briefly, compound was agitated for 30 hoursand sampled, centrifuged, and analyzed by UPLC. Solubility in SGF wasfound to be 1.05 mg/ml. The results from these studies indicate thatsolubility of Compound 2 is not significantly dependent on pH of themedia, but may have some increased solubility based on the presence ofbile salts.

Compounds of the invention were also tested for solubility in NormalRinger Solution. Briefly, compound solubility was determined bydissolving a standard range of volumes of 10 mM DMSO stock of compoundsin Normal Ringer Solution (145 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mMMgCl₂, 10 mM HEPES, 10 mM glucose; pH 7.4 at room temperature).Following vortex and incubation for 40 minutes at room temperature,solutions were filtered, quenched with acetonitrile, and analyzed byliquid chromatography. Solubility limits were determined by comparisonto a standard curve. The solubility limit was determined to be greaterthan 31.3 μM. Solubility is reported as “greater than” if the observedincrease between the last 2 dilutions tested is greater than 2-fold.Table 5 shows the values achieved for the compounds tested.

TABLE 5 Solubility of compounds of Formula (I) in Normal Ringer SolutionCompound Solubility (nM) 1 >14300 2 ~32400 4 >31300 5 4370 6 ~2300013 >77600 15 16300 19 >18200 16 >47000 26 >15100

Binding of Compound 2 to human, rat, dog, and cynomolgus monkey plasmaproteins was tested using an equilibrium dialysis approach. With thismethod, free compound is separated from protein-bound compound bydialysis across a semi-permeable membrane. At a concentration of 1 μM,Compound 2 demonstrated 98.5% binding to human, 95.3% binding to rat,94.5% to dog and 98.0% to cynomolgus monkey plasma proteins (Table 6).

The protein binding was tested at a concentration of 1 μM. Pooled human,cynomolgus monkey, dog, and rat K2EDTA plasma was thawed and centrifugedat 2000×g for 10 minutes at 4° C. to remove particulates; any lipid onthe top of the supernatant was also removed by aspiration. The plasmawas warmed to 37° C. for 10 minutes before use. Test compounds werespiked into 2 ml plasma in a polypropylene plate to a finalconcentration of 1 μM. Triplicate 400 μl aliquots of spiked plasma weretransferred into the Thermo RED dialysis units and dialyzed against 600μl of PBS buffer. The RED devices were incubated at 37° C. with gentleshaking using a Boekel Jitterbug 130000; the plates were also protectedfrom light. After 6 hours dialysis, triplicate 50 μl aliquots wereremoved from the RED plate, matrix-matched with either PBS buffer orblank plasma, as appropriate, and quenched with 4 volumes of ACNcontaining an internal standard. The extracted samples were thencentrifuged at 2000×g for 5 minutes at 4° C. The supernatant (50 μl) wasremoved and diluted with 100 μl water prior to LC/MS/MS bioanalysis.

TABLE 6 Comparison of binding of Compound 2 and warfarin to plasmaproteins Compound ID Species Mean % Bound ± SD Compound 2 Human 98.5 ±0.1 Compound 2 Sprague dawley rat 95.3 ± 0.8 Compound 2 Beagle dog 94.5± 0.5 Compound 2 Cynomolgus monkey 98.0 ± 0.1 Controls Warfarin Human99.1 ± 0.2 Warfarin Sprague dawley rat 99.1 ± 0.1 Warfarin Beagle dog93.5 ± 0.4 Warfarin Cynomolgus monkey 98.9 ± 0.3

The change in IC₅₀ block in the presence of albumin and plasma was alsostudied. It is assumed that the pharmacological effect of a drugcorrelates to unbound plasma levels, which is known as the “free drughypothesis”. The aim of this study was to estimate the change in IC₅₀for block of human TRPA1 (hTRPA1) by Compound 2 in the presence ofphysiologically relevant concentrations of albumin and plasma (see Table7). The human form of TRPA1 was used to assess protein binding in allspecies due to technical issues. The whole-cell patch clamp technique asdescribed in del Camino, D. et al. J Neurosci 74 (2010) 30:15165 wasemployed to measure current through hTRPA1 upon activation by allylisothiocyanate (AITC), the active ingredient in mustard oil, in thepresence of 1% (w/v) serum albumin or 25% (v/v) plasma from variousspecies including human plasma (hPlasma), human serum albumin (HSA), ratplasma (rPlasma), rat serum albumin (RSA), dog plasma, and sheep serumalbumin (sheepSA). Compound 2 was sub-diluted from a 10 mM stock in DMSOto 10 and 100 μM in DMSO, then diluted into Ringer solution at theconcentrations referenced in Table 7 and Table 8.

The subsequent introduction of Compound 2 resulted in dose-dependent andreversible blockade of hTRPA1. In the presence of 25% (v/v) human plasma(hPlasma) hTRPA1 currents were blocked with an IC₅₀ of 95±2 nM, which is14-fold higher than the IC₅₀ in the absence of serum (see Table 8).Experiments performed on hTRPA1 in the presence of rat plasma (rPlasma)and rat serum albumin (RSA) yielded a potency of block by Compound 2 of68±8 nM and 95±9 nM, respectively indicating a degree of protein bindingsimilar to that observed with human plasma. The IC₅₀ for block byCompound 2 was somewhat higher in the presence of dog plasma (221±54nM). In the presence of 1% (w/v) sheep serum albumin (SheepSA) hTRPA1currents were blocked with an IC₅₀ of 70±10 nM. Both block with Compound2 and reversal upon washout were complete within 2-3 minutes. Theseexperiments indicate that Compound 2 will exert pharmacological effectsat lower plasma levels than previously identified compounds, becausemore free drug is available to interact with the target.

Based on the shift in IC₅₀ observes in the presence of plasma, it may beconcluded that Compound 2 displays significant binding to plasmaproteins across the four species tested in the range of 90-97%. Thisrepresents an improvement over past compounds of similar potency.

TABLE 7 Compound 2 hTRPA1 IC₅₀ determination in albumin and plasma IonTested Current IC₅₀ Fold Test Channel Concs. (nm) n Activation (nM)Shift IC₅₀ + hPlasma hTRPA1 32, 100, 320 3 20 μM AITC 95 ± 2 14X IC₅₀ +rPlasma hTRPA1 32, 100, 320, 4 20 μM AITC 68 ± 8 10X 1000 IC₅₀ + RSAhTRPA1 32, 100, 320 3 20 μM AITC 95 ± 9 14X IC₅₀ + dog Plasma hTRPA1 32,100, 320, 4 20 μM AITC 221 ± 54 32X 1000, 3200 IC₅₀ + SheepSA hTRPA1 32,100, 320 3 20 μM AITC 70 ± 10 10X

TABLE 8 Compound 2 IC₅₀ determination for hTRPA1 from various mammaliansources Tested concs. Current IC₅₀ Inward Test Ion Channel Species (nm)Activation n current (nM) IC₅₀ hTRPA1 Human 1, 3.2, 10 μM AITC 13 6.9 ±3.1 10, 32, 100 IC₅₀ rTRPA1 Rat 10, 32, 10 μM AITC 3 22.9 ± 4.6 100 IC₅₀dogTRPA1 Dog 3.2, 10, 10 μM AITC 4 23.5 ± 6.4 32, 100 IC₅₀ mTRPA1 Mouse10, 32, 10 μM AITC 3 26.6 ± 3.3 100, 320 IC₅₀ sheepTRPA1 Sheep 1000, 20μM AITC 3 3280 ± 640 3200, 10000

Metabolic Stability

Metabolic stability of the compounds of Formula (I) was determined bystandard liver microsome assays. Briefly, metabolic stability was testedby adding the compound to be tested dissolved in DMSO to human, dog, orrat liver microsomes. Assays were run with a starting concentration of 1μM test compound. The reaction was initiated by addition of nicotinamideadenine dinucleotide phosphate-oxidase (NADPH) regeneration componentsat 37° C., at which time an aliquot was immediately quenched in anice-cold acetonitrile/methanol/water solution. Reaction mixture wasincubated at 37° C. on a shaker, and additional aliquots were taken at7, 15, 30 and 60 minutes. Following quench and centrifugation, sampleswere analyzed on HPLC/MS/MS. Results are shown in Table 9, Table 10, andTable 11 below.

TABLE 9 Half life and hepatic clearance of compounds in human livermicrosomes Human Liver Microsomes Human Liver Microsomes HepaticClearance Compound Half-Life (min) (mL/min/kg) 1 9 16 2 43 8 4 19.8 537.8 6 27.4 8 38.4 13 >60 15 88.8 17 8 16 18 12 15 19 50 9 21 30 10.5 2226 12 23 4 18 25 43 8 16 52.4 26 >120

TABLE 10 Half life and hepatic clearance of compounds in dog livermicrosomes Female Female Male Half- Male Half-Life Hepatic HepaticHepatic Life Half-Life both sexes Clearance Clearance Clearance Compound(min) (min) (min) (mL/min/kg) (mL/min/kg) (mL/min/kg) 1 18 10 18 22 222.9 22.9 5 >120 19 44 11 21 66 8

TABLE 11 Half life and hepatic clearance of compounds in rat livermicrosomes Female Female Male Half- Male Half-Life Hepatic HepaticHepatic Life Half-Life Both sexes Clearance Clearance Clearance Compound(min) (min) (min) (mL/min/kg) (mL/min/kg) (mL/min/kg) 1 46 22 39.2 20 292 786.6667 >60 2 4 72.6 5 113 6 118 8 115 13 60 15 72.7 17 22 18 21.8 2419 36 63.8 25 21 16 34 11

Bioavailability

Early bioavailability studies in rat were conducted with solutions ofthe compounds of the invention. Compounds were delivered via oraladministration as a solution in an appropriate excipient. Exampleformulations include, but are not limited to: 4% DMSO, 10% SolutolHS-15, and 86% water or 4% DMSO, 5% Tween, 25% Cremophor EL. Targetconcentrations were typically 1 mg/mL, and administered via oral gavageto non-fasted rats. The absolute bioavailability is the dose-correctedarea under the curve (AUC) non-intravenous divided by the dose correctedAUC intravenous. The formula for calculating F for a drug administeredby the oral route (PO) is given below:

% F=AUC PO×Dose IV/AUC IV×Dose PO

The respective bioavailability for rats for the compounds tested isshown in Table 12.

TABLE 12 Bioavailability of compounds in non-fasted rats Rat Compound %F 1 46 2 100 4 100 15 35 19 77

Additional studies were conducted with micronized Compound 2 in rat,dogs (beagles), and cynomolgus monkeys. Animals received a 10 mg/kg doseoral dose of compound formulated as a suspension in 0.5% methylcellulose in water for injection at a target concentration of 1 mg/mL,and administered a dose volume of 10 mL/kg by oral gavage in rats orfasted dogs and via oral delivery in fasted cynomolgus monkeys. The % Ffor the rats in these studies was 85%, 36% for the dogs, and 19% for themonkeys. FIG. 11 depicts the pharmacokinetic profile for these species.

Several in vivo studies were performed to characterize thebioavailability of Compound 2. In Sprague-Dawley rats, a single oraldose of 10 mg/kg to 1000 mg/kg of Compound 2 were compared in a seriesof experiments. Compound 2 used in these studies was recrystallized fromethanol and micronized. Exposures based on area under the curve (AUC)and maximum plasma concentration (C_(max)) increased with doses up to1000 mg/kg.

Three studies were conducted in dogs (beagles). In one study, threefasted dogs were administered 10 mg/kg of a suspension of micronizedCompound 2 by gavage. Blood samples were collected from these dogsbefore dosing and at 0.25, 0.50, 1, 2, 4, 6, 8, 12, and 24 hourspost-administration. Blood samples were analyzed to determine plasmalevels of Compound 2 by LC/MS/MS. In subsequent studies, pharmacokinetic(PK) parameters were determined following a single oral dose ofsuspensions of micronized Compound 2 (recrystallized from ethanol)administered to fasted dogs at dose levels of 10, 100, 300, 600, or 1000mg/kg. Blood samples were taken before dosing and at 0.5, 1, 2, 4, 8,12, 24 and 48 hours post-dosing and analyzed to determine plasma levelsof Compound 2. Exposures on an AUC and C_(max) basis increased withdoses up to 600 mg/kg.

Three studies were conducted in fasted cynomolgus monkeys to determinethe PK profile and bioavailability of a suspension formulation ofCompound 2. Three monkeys were administered a suspension containing 10mg/kg micronized Compound 2 by oral gavage. Monkeys were bled beforedosing and at 0.25, 0.50, 1, 2, 4, 6, 12, and 24 hours post-dosing.Compound 2 plasma levels were determined by LC/MS/MS. Additional studieswere conducted to evaluate the PK profile at higher dose levels ofCompound 2 administered to fasted monkeys as an oral suspension. Bloodsamples were collected prior to dosing and up to 24 hours post-dosing.An additional 48 hour blood sample was collected from monkeys dosed with300 mg/kg Compound 2. Plasma levels of Compound 2 were determined byLC/MS/MS. Exposures on an AUC and C_(max) basis increased with doses upto 1000 mg/kg.

Another study was conducted to compare the PK parameters of capsule andsuspension formulations of Compound 2. All monkeys were dosed with 250mg Compound 2. Two groups were dosed with the capsule formulation: onefasted and one allowed food ad libitum. The animals that received thesuspension formulation were dosed using a nasogastric tube and werefasted. As seen in FIG. 12 and summarized in Table 13 below, the capsuleformulation appeared to be associated with an increased bioavailabilitycompared to the suspension, although the numerical difference was small.When the PK profile of the capsule formulation was compared in bothfasted and fed monkeys, the bioavailability of Compound 2 capsules wasnumerically greater in fasted monkeys.

TABLE 13 Comparison of PK parameters of Compound 2: capsule andsuspension formulations Compound 2 Dose (mg) Compound 2 Formulation F(%) 250 Suspension (fasted) 7.8 250 Capsule (fasted) 13 250 Capsule(fed) 10

Example 33 Method for Measuring Inhibition of the TRPA1 Ion Channel

Compounds of Formula (I) inhibit the TRPA1 channel, as shown bymeasuring the in vitro inhibition of human TRPA1, provided in datatables shown in Table 14, using the procedure outlined in del Camino etal., J Neurosci (2010) 30(45):15165-15174, which is incorporated hereinby reference and summarized below. Data for TRPA1 inhibition wasobtained by this method for the indicated compounds of Formula (I), withthe relevant data included in Table 14 below. All currents were recordedin whole-cell configuration using EPC-9 and EPC-10 amplifiers andPatchmaster software (HEKA). Patch pipettes had a resistance of 1.5-3 Mand up to 75% of the series resistance was compensated. The standardpipette solution consisted of 140 mM CsAsp, 10 mM EGTA, 10 mM HEPES,2.27 mM, 20 MgCl₂, 1.91 mM CaCl₂, and up to 0.3 mM Na₂GTP, with pHadjusted to 7.2 with CsOH. In addition, a solution containing 145 mMCsCl, 10 mM HEPES, 10 mM EGTA, and up to 0.3 mM Na₂GTP and 1 mM MgCl₂(pH 7.2 adjusted with CsOH) can be used. The standard bath solutioncontained 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 4.5 mM KCl, 1 mMEGTA, 3 mM MgCl₂, with pH adjusted to 7.4 with NaOH. In some instances,2 mM CaCl₂ was added in place of EGTA and the concentration of MgCl₂ wasreduced to 1 mM.

Data were collected either by continuous recordings at −60 mV or byapplying voltage ramps from a holding potential of 0 mV every 4 s.Continuous recordings were collected at 400 Hz and digitally filteredoff-line at 10 Hz for presentation. Voltage ramps were applied from −100mV to 100 mV over the course of 400 ms, and data were collected at 10kHz and filtered at 30 2.9 kHz. Inward and outward currents wereanalyzed from the ramps at −80 and 80 mV, respectively. Liquid junctionpotential correction was not used.

Solutions were switched using a gravity-fed continuous focal perfusionsystem. To achieve rapid temperature changes, two temperature control,and perfusion systems were employed simultaneously. For temperaturesgreater than or equal to 22° C., a Warner Instruments bipolartemperature controller (TC-344B) and inline heater (SHM-8) were used.For temperatures below 22° C. a Warner Instruments temperaturecontroller (CL-100) and thermal cooling module (TCM-1) were used.Temperatures were confirmed using a thermistor (Warner Instruments,TA-29), with temperatures at the recorded cell estimated to be within+/−2° C. of those reported.

Table 14 shows data obtained from the in vitro assay described above.The antagonist effects of compounds of Formula (I) against human TRPA1(“hTRPA1”) in a whole cell patch configuration were evaluated using thein vitro assay protocol described above.

TABLE 14 Antagonist effects of Compounds of Formula (I) against humanTRPA1 hTRPA1 Compound (nM) 1 5.15 2 6.87 3 2290 4 11.3 5 663 6 29.7 7922 8 71.9 9 462 10 93.5 11 124 12 387 13 62.9 14 94.8 15 26.7 16 24.917 41.2 18 10.4 19 96.1 21 51 22 >3200 23 60.7 24 436 25 107 26 21.8 27255 28 65.9

Example 34 Effect on Cold Hypersensitivity

Embodiments of the invention may be efficacious in the treatment ofinflammatory pain. Compound 2 tested by the CFA-induced pain testmethod. Compound 2 was formulated as a clear solution in 4% DMSO, 10%Solutol, 86% DWI, pH 5.9 for oral administration (PO).

Briefly, the hind paw is sensitized to cold temperature (allodynic), byadministering 0.1 mL of Complete Freund's Adjuvant (CFA) is administeredto the right hind paw. Three days later, the time taken for the animalto lift its CFA-injected paw is recorded compared to its un-injectednormal left hind paw. Animals are placed on the surface of the coldplate (1° C.) and the operator stops testing at the instant when theanimal displays discomfort by flinching or lifting its paw from theplate (paw withdrawal latency, or PWL). To avoid tissue damage themaximum cut-off time is 5 minutes. Animals that are allodynic (averagePWL to the first three pain behaviors <150 seconds for the CFA-injectedhind paw: ˜≧50% difference between the normal and CFA-injected paw) areincluded in the study and subsequently randomized across treatmentgroups. The following day, the animals are dosed under blindedconditions. Following the 1-2 hour pre-treatment time, the post-dose PWLreadings are again taken. The efficacy of the drug treatment is assessedby comparing the PWL in the drug treatment animals to those animals thatreceive the vehicle.

As shown in FIG. 7 and Table 15, Compound 2 attenuated coldhypersensitivity after oral doses of 0.3 to 10 mg/kg. The positivecomparator TRPA1 antagonist Compound A also reduced coldhypersensitivity at a higher dose of 150 mg/kg delivered viaintraplantar injection. Importantly, the vehicle delivered orally (4%DMSO, 10% Solutol, 86% DWI) had no effect on paw withdrawal latency,compared to pre-administration baseline measurements.

TABLE 15 Attenuation of cold hypersensitivity by Compound 2 at varyingoral dosages Pre-RX Post RX Dosage of PWL PWL Compound 2 (seconds)(seconds) Vehicle PO (n = 10) 104.5 136.1 0.3 mpk PO (n = 10) 104.0197.7 1 mpk PO (n = 10) 103.7 243.0 3 mpk PO (n = 10) 103.9 249.6 10 mpkPO (n = 10) 104.0 237.3 Compound A @ 104.2 213.2 150 mpk IP (n = 10)

Table 16 summarizes the average plasma levels of Compound 2 and CompoundA. Approximately dose proportional exposures of Compound 2 were observedthroughout the dose range tested. Decreased plasma binding of Compound 2indicates an improved bioavailability of Compound 2 to the subjects overCompound A.

TABLE 16 Plasma levels of Compound 2 and Compound A Treatment DosePLASMA Group (mg/kg) Route (ng/mL) Compound 2 0.3 PO  70 ± 11 Compound 21 PO 265 ± 56 Compound 2 3 PO  800 ± 160 Compound 2 10 PO 2780 ± 425Compound A 150 IP  7830 ± 3970

There were no differences in behavior between the vehicle and treatmentgroups (see Table 17). However lethargy/slow movement was noted in 5/10animals treated with the positive comparator, Compound A, demonstratingthat Compound 2 does not induce a significant sedative effect.

TABLE 17 Examination of animal behavior upon administration of Compound2 # Animals with low Treatment Dose activity/slow movement Group (mg/kg)Route or frank lethargy* Vehicle — PO 2/10 Compound 2 0.3 PO 1/10Compound 2 1 PO 1/10 Compound 2 3 PO 2/10 Compound 2 10 PO 2/10 CompoundA 150 IP 5/10

Compound 4 was also tested using the methods disclosed. Compound 4 wasformulated as a suspension in 0.5% methylcellulose and administered atthe doses indicated in Table 18.

TABLE 18 Attenuation of cold hypersensitivity by Compound 4 at varyingoral dosages Pre-Rx Post-Rx PWL PWL PWL Change Treatment (seconds)(seconds) (seconds) Vehicle, PO (n = 10) 101.9 100.6 −1.3 Compound 4 1mg/kg, 101.7 250.3 148.6 PO (n = 10) Compound 4 3 mg/kg, 101.1 242.1140.9 PO (n = 10) Compound 4 10 mg/kg, 101.5 234.1 132.6 PO (n = 10)Compound A 150 mg/kg, 104.9 210.8 105.9 IP (n = 8)

In a further study, compounds of the invention were tested for efficacyat low doses for the treatment of inflammatory pain. Using the methodsdisclosed in above, Compound 2 was dosed at ranges of 0.1 to 1 mg/kg PO.The positive comparator TRPA1 antagonist Compound A was also tested at adose of 150 mg/kg IP. Compound 2 was formulated as a clear solution in4% DMSO, 10% Solutol, 86% DWI, pH 5.9 for oral administration (PO) at adose volume of 10 ml/kg. Oral drug delivery was accomplished using a20-gauge 1½″ oral gavage needle and a 5 cc syringe. Fed rats received asingle oral gavage of Compound 2 at 0.03, 0.1, 0.3, or 1 mg/kg orVehicle, 2 hours prior to testing

As seen in Table 19 and FIG. 13, Compound 2, when dosed at 0.1, 0.3, and1 mg/kg PO, showed a significant reversal of CFA-induced coldhypersensitivity, as assayed by measuring paw withdrawal latency. 0.03mg/kg dose levels did not exert a statistically significant effect. Thepositive comparator, the prototypic TRPA1 antagonist Compound A, alsoshowed a significant reversal of cold hypersensitivity when dosed at 150mg/kg IP.

TABLE 19 Reversal of CFA-induced cold hypersensitivity by Compound 2 PWLChange Dosage (Sec) SEM Vehicle PO (n = 10) 1.6 15.7 Compound 2 @ 0.03mpk PO (n = 10) 14.8 12.2 Compound 2 @ 0.1 mpk PO (n = 10) 43.1 16.4Compound 2 @ 0.3 mpk PO (n = 11) 74.1 18.1 Compound 2 @ 1 mpk PO (n =11) 104.8 24.7 Compound A @ 150 mpk IP (n = 10) 135.8 16.6

In summary, these studies suggest that compounds of the invention havethe potential to be efficacious in the treatment of inflammatory painfollowing oral administration.

Example 35 Formalin Model

Compound 2 was tested in the formalin-induced pain test reported byDubuisson et al., Pain (1977) December; 4(2):161-74. Dubuisson et aldescribe a method for assessing pain and analgesia in rats and cats.Briefly, dilute formalin (50 μL of 3% formalin) is injected into theplantar surface of the hind paw of a rat. The animal is promptlyreturned to an observation arena (standard Plexiglass rat cage), atwhich point a trained observer records the time the animal spendsexhibiting pain behaviors (flinching, licking, biting of the injectedpaw/leg) in two distinct phases. The initial phase (Phase I: 0-5 min) isthought to have a significant component that is dependent upon directactivation of afferent fibers by formalin and functional TRPA1 (McNamaraet al., 2007). The individual responsible for counting the painbehaviors in a particular study is blinded to the treatment groups.

Investigators studied oral administration of Compound 2 at 1, 3 and 10mg/kg on pain behaviors in the formalin model in the rat. Compound 2 wasprepared as solutions in 4% DMSO, 10% Solutol HS15, 86% WFI. Animalswere dosed orally with Vehicle (4% DMSO, 10% Solutol, 86% WFI), orCompound 2 at 1, 3 or 10 mg/kg one hour prior to intraplantar formalin.FIG. 14 shows the duration of pain behaviors observed in the first twominutes (Left) or the duration of pain behaviors during the entire studyperiod; five minutes (Right). (n=8 per group) (*=p<0.05, **=p<0.01, ***p<0.001: 1-tailed T-test)

Oral administration of Compound 2 significantly reduced the nociceptiveresponses in Phase 1 of the formalin model at 3 and 10 mg/kg as seen inTable 20 and FIG. 14. Compared to vehicle treated animals, animalstreated with Compound 2 at 1 mg/kg resulted in a ˜14% decrease in theduration of pain behaviors from 0-2 minutes following intraplantarformalin, although this reduction was not statistically significant. Atdoses of 3 and 10 mg/kg PO, Compound 2 resulted in a significantdecrease in formalin-induced pain behaviors from 0-2 minutes by ˜72% and˜89%, respectively, compared to vehicle treated animals.

A similar reduction in the duration of pain behaviors was also observedwith Compound 2 from 0-5 minutes post-formalin administration. At 1mg/kg PO, Compound 2 reduced pain behaviors by ˜14%, but did not reachstatistical significance compared to vehicle treated animals. At 3 and10 mg/kg PO, Compound 2 significantly reduced the duration offormalin-induced pain behaviors by ˜46% and ˜60%, respectively.

TABLE 20 Dose response of Compound 2 administered orally using theformalin model Duration Duration (seconds) (seconds) Dose 0-2 min 0-5min Vehicle PO (n = 8) 84.50 182.00 Compound 2 @ 1 mpk PO (n = 8) 73.00157.38 Compound 2 @ 3 mpk PO (n = 8) 23.25 99.00 Compound 2 @ 10 mpk PO(n = 8) 9.25 73.50

A reduction in the duration of pain behaviors was also observed withCompound 1 from 0-5 minutes post-formalin administration. At 1 mg/kg and3 mg/kg delivered intravenously to rats, Compound 1 reduced the durationof formalin-induced pain behaviors as shown in Table 21 and FIG. 15.

TABLE 21 Dose response of Compound 1 administered intravenously usingthe formalin model Duration Duration (seconds) (seconds) Dose 0-2 min0-5 min Vehicle (n = 8) 71.00 160.75 Compound 1 @ 1 mg/kg IV (n = 8)35.50 153.25 Compound 1 @ 3 mg/kg IV (n = 8) 6.25 111.25

A reduction in the duration of pain behaviors was also observed withCompound 4 from 0-5 minutes post-formalin administration. Using themethods described above for the formalin assay, Compound 4 wasformulated as a solution in 4% DMSO; 5% Tween-80; 20% Cremophor EL; and71% WFI and administered by oral gavage to rats. Compound 4 reduced theduration of formalin-induced pain behaviors as shown in Table 22.

TABLE 22 Dose response of Compound 4 administered orally using theformalin model Duration Duration (seconds) (seconds) Dose 0-2 min 0-5min Vehicle PO (n = 8) 91.50 237.25 Compound 4 @1 mpk PO (n = 8) 76.13197.88 Compound 4 @3 mpk PO (n = 8) 45.25 175.88 Compound 4 @10 mpk PO(n = 8) 47.13 191.00

Using the methods described above for the formalin assay, Compound 4 wasformulated as a suspension in 0.5% methylcellulose and administered byoral gavage to rats. Compound 4 reduced the duration of formalin-inducedpain behaviors as shown in Table 23.

TABLE 23 Dose response of Compound 4 administered orally using theformalin model Duration Duration (seconds) (seconds) Dose 0-2 min 0-5min Vehicle PO (n = 8) 94.25 192.75 Compound 4 @ 1 mpk PO (n = 8) 80.38186.25 Compound 4 @ 3 mpk PO (n = 8) 51.13 163.25 Compound 4 @ 10 mpk PO(n = 8) 38.13 144.25

The persistence of the response was also studied. Compound 2 wasprepared as a solution in 4% DMSO, 10% Solutol HS15, and 86% WFI. Ratswere treated with 10 mg/kg of oral doses of Compound 2 or with thevehicle (PO). FIG. 8 shows that pre-treatment with oral dose formulationof Compound 2 at 10 mg/kg PO from 30 minutes to 6 hours prior toformalin injection significantly decreased the duration offormalin-mediated pain behaviors.

Compared to vehicle treated animals, 15 minute to 6 hour pre-treatmentwith oral Compound 2 at 10 mg/kg PO resulted in an ˜30-87% decrease inthe duration of formalin-induced pain behaviors 0-2 minutes followingformalin injection, with the maximum decrease in pain behavior observedin the 2 hour pre-treatment group as shown in Table 24.

TABLE 24 Duration of pain response upon oral administration of Compound2 using the formalin model Duration Duration (seconds) (seconds) Dose0-2 min 0-5 min Vehicle PO (n = 8) 89.63 179.73 15 Min (n = 8) 82.50185.43 30 Min (n = 8) 62.50 181.40 1 Hr (n = 8) 14.50 146.14 2 Hr (n =8) 12.00 118.10 4 Hr (n = 8) 19.00 135.84 6 Hr (n = 8) 22.75 156.61 24Hr (n = 8) 96.63 197.15

Compound 2 was also tested in the sheep model of allergicbronchoconstriction and airway hyperresponsiveness according to themethods disclosed in Abraham, W. M Palm Pharmacol Ther (2008)21:743-754. Allergic sheep challenged with Ascaris suum show asubstantial, biphasic increase in pulmonary resistance (RL). The firstfour hours are considered the early asthmatic response (EAR); the nextfour hours (hours 4-8) are considered to be the late asthmatic response(LAR). To assess airway responsiveness (AHR), the cumulative carbacholdose in breath units that increased pulmonary resistance 400% over thepost-buffer value (PC 400) was calculated from the dose response curve.One breath unit was defined as one breath of a 1% w/v carbacholsolution. A pre-challenge PC 400 was obtained 1-3 days before the startof dosing.

Compound 2 was formulated as a micronized powder suspended in 0.5%methylcellulose at a concentration of 6 mg/ml and administered orally ata dose of 30 mg/kg once a day for 4 days, at the same approximate timeeach day. Sheep were given 30 mg/kg Compound 2 orally daily four days.Two hours after the final dose of Compound 2, the sheep were subjectedto an allergen (Ascaris) challenge. Each sheep was restrained in a proneposition and its head was immobilized prior to topical anesthesia of thenasal passages. A balloon catheter was advanced through one nostril intothe lower esophagus. Each sheep was intubated with a cuffed endotrachealtube through the other nostril. Tracheal and pleural pressures weredetermined using the endotracheal tube and balloon catheter,respectively. The trans-pulmonary pressure, i.e., the difference betweenthe tracheal and pleural pressures, was measured using a differentialpressure transducer catheter system. R_(L) was determined by connectingthe distal end of the endotracheal tube to a pneumotachograph. Data werecollected from five to ten breaths to a computer and used to calculateR_(L). Data from the same sheep challenged with Ascaris prior to theinitiation of Compound 2 treatment were used to establish baselinevalues. Monitoring conditions for the control and drug trials wereidentical.

On the day of challenge and two hours after the final dose of Compound2, an aerosol of Ascaris suum (82,000 protein nitrogen units/mL) wasgenerated using a nebulizer and delivered to the sheep using a Harvardrespirator. R_(L) was determined one hour prior to challenge,immediately following Ascaris challenge, and hourly thereafter for eighthours. Challenge to 4 hours is considered the EAR while 4 to 8 hours isconsidered the LAR.

Sheep were also challenged with aerosolized carbachol, a cholinergicagonist that has a negative impact on AHR. The carbachol concentrationin breath units that increased RL 400% (PC400) determinations wereperformed 24 hours following Ascaris challenge without Compound 2 dosing(historical baseline) or 24 hours after administration of the final doseof Compound 2.

FIG. 16 shows the antigen-induced responses in sheep at baseline(control) levels and following treatment with Compound 2 (30 mg/kg).Compound 2 did not affect the peak early airway responses. However, itdramatically attenuated the late airway responses (85% protection). Inthe control trial the average late airway responses was 126±4.7%,whereas in the treatment trial the average LAR was only 19±2.3%(P=0.002).

FIG. 17 further shows the effect of Compound 2 (30 mg/kg) on PC400, ameasurement of airway hyperresponsiveness representing the concentrationof carbachol that induces a 400% increase in lung resistance.

In summary, treatment with Compound 2 reduced the airwayhyperresponsiveness to levels similar to those observed in sheep thatwere not challenged with Ascaris suum.

Example 36 Pharmaceutical Profile

Compounds of the invention may not have significant drug/druginteractions, making administration preferable to patients takingmultiple medications.

The ability of Compound 2 to inhibit human CYP450 enzymes was evaluated.Compound 1 and Compound 2 were tested in standard P450 Cyp-inhibitionluminescent assay. Results are shown in Table 25 below.

TABLE 25 Inhibition of CYP450 enzymes by Compounds 1 and 2 CYP 1A2: CYP2C19: CYP 2C9: CYP 2D6: CYP 3A4: Com- % % % % % pound InhibitionInhibition Inhibition Inhibition Inhibition 1 4.2 45.9 71.4 9 26.9 20.299 29.1 39.6 6.3 2.7

Compound 2 achieved a maximum block of human CYP450 enzymes of up to 37%at 10 μM for the seven isozymes tested; these values indicate thatcalculated IC₅₀ values would be >10 μM (Table 26).

CYP450 reaction phenotyping of Compound 2 was conducted by incubatingthe test article with human liver microsomes in the presence and absenceof selective CYP450 inhibitors. The metabolic half-lives were notsignificantly affected by any of the CYP450 inhibitors except forketoconazole, indicating that the in vitro metabolism of Compound 2involved mainly the CYP3A4 isozymes (FIG. 9).

TABLE 26 CYP450 inhibition at 10 μM by Compound 2 and appropriatereference compounds Mean % CYP450 CYP450 Inhibition at Compound IDIsozyme Substrate 10 μM Compound 2 1A2 Phenacetin 9.0 Compound 2 2B6Buproprion 15.1 Compound 2 2C8 Amodiaquine 3.2 Compound 2 2C9 Diclofenac22.0 Compound 2 2C19 Mephenytoin 37.1 Compound 2 2D6 Dextromethorphan9.5 Compound 2 3A4 Midazolam 0.0 Compound 2 3A4 Testosterone 2.8Controls: Fluvoxamine 1A2 Phenacetin 96.6 Ticlopidine 2B6 Buproprion98.2 Quercetin 2C8 Amodiaquine 61.5 Sulfaphenazole 2C9 Diclofenac 95.3Omeprazole 2C19 Mephenytoin 68.2 Quinidine* 2D6 Dextromethorphan 55.8Ketoconazole 3A4 Midazolam 99.2 Ketoconazole 3A4 Testosterone 98.8 *1 μMAssay Concentration

Example 37 Hepatotoxicity Safety Profile in Dogs

Compound 2 in vehicle (0.5% methylcellulose [400 cps] in deionizedwater) was administered orally once daily for five consecutive days bygavage once to 3 groups of non-naïve male and female beagle dogs. Eachgroup received one dose level. Dose levels were 300, 600, and 1000 mg/kgfor each group. A concurrent control group received the vehicle on acomparable regimen. The dose volume was 10 ml/kg for all groups.Hepatoxicity was measured via the serum biomarkers of alanineaminotransferease [ALT], aspartate aminotransferase [AST], alkalinephosphastase [ALP] and gamma-glutamyl transferase [GGT] which representhepatotoxicity or bile duct injury. Table 27 shows that Compound 2 inthe dogs at each dose level indicated did not elevate the serumbiomarkers up to and including a dose of 300 mg/kg.

TABLE 27 Hepatotoxicity safety profile of Compound 2 in beagle dogsSerum 0 mg/kg 30 mg/kg 100 mg/kg 300 mg/kg Biomarker (U/L) (U/L) (U/L)(U/L) ALP d-5 93.0 93.0 120.0 118.0 ALP d5 102.0 143.0 141.0 101.0 ALTd-5 24.0 19.0 21.0 28.0 ALT d5 21.0 20.0 19.0 21.0 AST d-5 23.0 20.020.0 22.0 AST d5 30.0 22.0 21.0 22.0 GGT d-5 0.0 0.0 0.0 0.0 GGT d5 0.00.0 0.5 0.0 n 1 2 2 2

FIG. 18 further demonstrates that Compound 2 does not significantlyelevate serum biomarker levels above normal ranges. FIG. 19 demonstratesthat Compound 2 did not significantly elevate serum biomarker levelsabove base line measurements as demonstrated by a % difference over thevehicle.

Example 38 Hepatotoxicity Safety Profile in Rats

Compound 2 in the vehicle (0.5% methylcellulose [400 cps]) wasadministered orally by gavage once daily for a minimum of 28 consecutivedays to 3 groups of sprague-dawley rats from Charles River Laboratories.Each group received one dosage level. Dosage levels were 30, 100, and300 mg/kg/day for each group. Concurrent control groups received thevehicle on a comparable regimen. The dose volume was 10 ml/kg for allgroups. Hepatoxicity was measured via the serum biomarkers of alanineaminotransferease [ALT], aspartate aminotransferase [AST], alkalinephosphastase [ALP] and gamma-glutamyl transferase [GGT] which representhepatotoxicity or bile duct injury. Table 28 shows that Compound 2 inthe rats as each dose level indicated did not elevate the serumbiomarkers up to and including a dose of 300 mg/kg.

TABLE 28 Hepatotoxicity safety profile of Compound 2 in rats Serum Doseof Compound 2 (U/L) Biomarker 0 mg/kg 30 mg/kg 100 mg/kg 300 mg/kg ALPd28 186.0 168.0 185.0 163.0 ALT d28 36.0 33.0 34.0 43.0 AST d28 116.098.0 100.0 115.0 GGT d28 0.0 0.0 0.0 0.0 n 10.0 10.0 10.0 10.0

FIG. 20 further demonstrates that Compound 2 does not significantlyelevate levels above normal ranges. FIG. 21 demonstrates that Compound 2did not significantly elevate levels above base line measurements asdemonstrated by a % difference over the vehicle.

Example 39 Hepatotoxicity Safety Profile in Monkeys

Compound 2 in the vehicle (0.5% methylcellulose, 400 cps) wasadministered via nasogastric intubation once daily for 28 or 29consecutive days to 4 groups of cynomolgus monkeys. Each group receivedone dose level. Dosage levels were 10, 30, 100, and 300 mg/kg/day pergroup. A concurrent control group received the vehicle on a comparableregimen. The dosage volume was 10 ml/kg for all groups. Hepatoxicity wasmeasured via the serum biomarkers of alanine aminotransferease [ALT],aspartate aminotransferase [AST], alkaline phosphastase [ALP] andgamma-glutamyl transferase [GGT] which represent hepatotoxicity or bileduct injury. Table 29 shows that Compound 2 in the monkeys at each doselevel indicated did not elevate the serum biomarkers up to and includinga dose of 300 mg/kg.

TABLE 29 Hepatotoxicity safety profile of Compound 2 in cynomolgusmonkeys Serum Dosage of Compound 2 (U/L) Biomarker 0 mg/kg 10 mg/kg 30mg/kg 100 mg/kg 300 mg/kg ALP d28 565.0 744.0 461.0 532.0 441.0 ALT d2859.0 87.0 50.0 61.0 67.0 AST d28 80.0 126.0 72.0 70.0 134.0 GGT d28 67.664.0 49.0 53.7 54.8 n 5.0 3.0 3.0 3.0 5.0

FIG. 22 further demonstrates that Compound 2 does not significantlyelevate levels above normal ranges. FIG. 23 demonstrates that Compound 2did not significantly elevate levels above base line measurements asdemonstrated by a % difference over the vehicle.

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A compound of the Formula (I) or a pharmaceutically acceptable saltthereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, optionally substitutedwith one or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano.
 2. The compound accordingto the preceding claim, wherein R¹ is C₁-C₆ alkyl.
 3. The compound ofclaim 2, wherein R¹ is —CH₃.
 4. The compound of claim 1, wherein R¹ isH.
 5. The compound of claim 1, wherein R² is H.
 6. The compound of claim1, wherein R² is C₁-C₆ alkyl.
 7. The compound of claim 6, wherein R² is—CH₃, —CD₃, or —CHF₂.
 8. The compound of claim 1, wherein each of R¹ andR² is independently C₁-C₆ alkyl.
 9. The compound of claim 8, whereineach of R¹ and R² is independently —CH₃.
 10. The compound of claim 1,wherein each of R¹ and R² is independently —CH₃ and R³ is H.
 11. Thecompound of claim 1, wherein R³ is H.
 12. The compound of claim 1,wherein R³ is C₁-C₆ alkyl.
 13. The compound of claim 12, wherein R³ is—CH₃.
 14. The compound of claim 1, wherein each of R¹, R², and R³ isindependently C₁-C₆ alkyl.
 15. The compound of claim 14, wherein each ofR¹, R² and R³ is independently —CH₃.
 16. The compound of claim 1,wherein the compound is of the Formula (Ia):

or a pharmaceutically acceptable salt thereof.
 17. The compound of claim1, wherein the compound is of the Formula (Ib):

or a pharmaceutically acceptable salt thereof.
 18. The compound of claim1, wherein R⁴ is heterocyclyl.
 19. The compound of claim 18, wherein theheterocyclyl is a 4 to 8-membered ring.
 20. The compound of claim 18,wherein the heterocyclyl is linked through a nitrogen atom.
 21. Thecompound of claim 18, wherein R⁴ is substituted heterocyclyl.
 22. Thecompound of claim 21, wherein R⁴ is selected from the group:


23. The compound of claim 22, wherein R⁴ is selected from the group:

and m is
 1. 24. The compound of claim 23, wherein R⁴ is selected fromthe group:


25. The compound of claim 22, wherein R⁴ is selected from the group:

and m is
 1. 26. The compound of claim 25, wherein R⁴ is selected fromthe group:


27. The compound of claim 1, wherein m is
 1. 28. The compound of claim1, wherein m is
 0. 29. The compound of claim 1, wherein R⁶ is alkyl,haloalkyl, or cyano.
 30. The compound of claim 29, wherein R⁶ is alkylor haloalkyl.
 31. The compound of claim 30, wherein R⁶ is —CF₃.
 32. Thecompound of claim 20, wherein R⁴ is selected from the group:


33. The compound of claim 1, wherein the compound of Formula (I) is ofthe Formula (II):

wherein: n is an integer from 0 to 4; and m is selected from an integerfrom 0 to
 4. 34. The compound of claim 1, wherein the compound ofFormula (I) is of the Formula (ha):

wherein: n is an integer from 0 to 4; and m is selected from an integerfrom 0 to
 4. 35. The compound of claim 1, wherein the compound ofFormula (I) is of the Formula (IIb):

wherein: n is an integer from 0 to 4; and m is selected from an integerfrom 0 to
 4. 36. The compound of claim 1, wherein the compound isselected from the following group:

or a pharmaceutically acceptable salt thereof.
 37. The compound of claim1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 38. The compound of claim37, wherein a solid crystalline form of the compound has an X-ray powderdiffraction pattern comprising characteristic peaks, expressed in termsof 2θ, at about 7.67°, about 12.52°, about 13.49°, and about 19.31°. 39.The compound of claim 37, wherein a solid crystalline form of thecompound has an X-ray powder diffraction pattern comprisingcharacteristic peaks, expressed in terms of 2θ, at about 9.78°, about12.98°, about 19.20°, and about 19.67°.
 40. The compound of claim 1,wherein a solid crystalline form of the compound has a melting point ofgreater than or equal to about 150° C.
 41. The compound of claim 1,wherein a solid crystalline form of the compound has a melting point inthe range of about 180° C. to about 205° C.
 42. The compound of claim 1,wherein a solid crystalline form of the compound has a melting point inthe range of about 190° C. to about 200° C.
 43. A purifiedpharmaceutical preparation comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano.
 44. The preparation of thepreceding claim, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 45. The preparation ofclaim 44, wherein the preparation comprises a diastereomeric excess ofgreater than or equal to about 99%.
 46. The preparation of claim 44,wherein the preparation has a moisture content of less than or equal toabout 0.1%.
 47. A pharmaceutical composition comprising a compound ofFormula (I) or a pharmaceutically acceptable salt thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano.
 48. A method for treatinga TRPA1 mediated disorder in a subject, the method comprisingadministering an effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof, to a subject in need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 49. A method for treating pain in a subject, the methodcomprising administering an effective amount of a compound of Formula(I), or a pharmaceutically acceptable salt thereof, to a subject in needthereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 50. The method of claim 49, wherein the pain is neuropathicpain.
 51. The method of claim 49, wherein the pain is inflammatory pain.52. The method of claim 49, wherein the pain is PDN or CIPN.
 53. Themethod of claim 49, wherein the pain is visceral pain.
 54. The method ofclaim 49, wherein the pain is selected from the group: cancer pain, burnpain, oral pain, crush and injury-induced pain, incisional pain, bonepain, sickle cell disease pain, fibromyalgia and musculoskeletal pain.55. The method of claim 49, wherein the pain is from hyperalgesia orallodynia.
 56. A method for treating inflammatory disease in a subject,the method comprising administering an effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, to a subjectin need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano.
 57. A method for treatingneuropathy in a subject, the method comprising administering aneffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, to a subject in need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 58. The method of claim 57, wherein the neuropathy is fromdiabetes, chemical injury, chemotherapy, and or trauma.
 59. A method fortreating a dermatological disorder in a subject, the method comprisingadministering an effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof, to a subject in need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 60. The method of claim 59, wherein the dermatological disorderis selected from atopic dermatitis, acute pruritus, psoriasis, hives,eczema, dyshidrotic eczema, mouth ulcers, and diaper rash.
 61. A methodfor treating a pulmonary disease in a subject, the method comprisingadministering an effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof, to a subject in need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 62. The method of claim 61, wherein the pulmonary disease is anobstructive disease.
 63. The method of claim 61, wherein the pulmonarydisease is chronic obstructive pulmonary disease or asthma.
 64. A methodfor treating cough in a subject, the method comprising administering aneffective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, to a subject in need thereof:

wherein: R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R² is H,C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted withone or more R⁵ groups; R³ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴ is halo, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido,thioyl, sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl, optionallysubstituted at one or more positions with 1-4 R⁶ groups; R⁵ isindependently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy,amino, amido, phosphonate, carboxyl, ether, alkylthio, haloalkyl, andcyano; and R⁶ is independently H, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, alkehyde, ester, heterocycle, an aromaticor heteroaromatic ring, haloalkyl, and cyano, to thereby treat thesubject.
 65. The method of claim 64, wherein the cough isallergy-induced cough.